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Theory and simulation of self-cycling fermentation:

a population balance approach

by

Francois Godin

Department ofChemical Engineering

McGill University, Montreal

A thesis submitted ta the Faculty ofGraduate Studies and Research in partial fulfillment of
the requirements ofthe Degree ofMaster of Engineering

McGill University

August, 1997

Francois Godin 1997

1+1

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ABSTRACT
Self-cycling fermentation (SCf) is a technique in which sequential batch
fermentations are performed using a computerized feedback control scheme. In this
method, halfof the reactor volume is periodically removed and replaced by fresh medium.
This results in very stable and repeatable growth cycles and synchronized cell cultures.
In this wode, two segregated microbial population balance models are developed

and used to simulate SCF. Cell age and cell mass distnDution models are bath used to
study the behavior of microorganisms in various systems. One example is the study of
autonomous oscillations and partial synchronization in cultures of Saccharomyces

cerevisiae. However, when the cell age and cell mass models are compared for the
modeDg of cell synchrony in SCF, two contrasting population profiles arise from the
simulations.
The SCF technique with its existing data can be used as a powerful tool to test and
validate models of microbial systems. When used ta simulate SCF, the cell age model was
able to predict cell synchrony, however, the ceU number profile obtained was remarkably
different than that observed in experiments. The cell mass model, as proposed by Eakman
et al. was able to capture the dissolved oxygen concentration, the limiting substrate

concentration, and the biomass concentration, but was not able ta describe the cell number
profile or the feature of cell synchrony. By introducing a feedback mechanism between
the critical division mass and the limiting substrate, ceU synchrony wu achieved and the
experimental cell profile was captured.

RSUM
Le procd de fermentation auto-cyclique (FAC) est une mthode de fermentation
squentielle discontinue qui est obtenue par l'entremise d'un contrle raction
informatis. Pour cette mthode, la moiti du volume du racteur est priodiquement
remplac par du bouillon de culture frais. On obtient des cycles de croissances stables et
rptitifs ainsi que ds cultures cellulaires synchronises.
Pour ce mmoire, deux modles cellulaires de population sgrgue sont
dveloppes et utilises pour simuler le FAC. Les modles de distribution cellulaires d'ge
et de masse sont tous deux utiliss pour tudier le comportement des microorganismes
dans diffrents systmes. Par exemple, les oscillations autonomes spontanes et les
synchronisations panielles dans les cultures de Saccharomyces cerev;siae ont dj t
tudies. Cependant, quand ces modles sont utiliss pour simuler le FAC, ils produisent
des rsultats diffrents.

La technique FAC avec les donnes existantes peut tre utilise comme un outil
puissant pour tester et valider les modles de systme microbiologique. Lorsqututilis
pour simuler le FAC, le modle dtge cellulaire pouvait prdire la synchronisation
cellulaire. Par contre le nombre de cellules en fonction du temps qui est obtenu est trs
diffrent de celui observ dans les exprimentations. Le modle de masse cellulaire,
comme propos par Eakman et al pouvait captur la concentration d'oxygne dissoute, la
concentration de substrat limit, et la concentration de biomasse sans pouvoir dcrire le
profile numrique ou la synchronisation cellulaire. En introduisant une relation entre la
masse critique de division et la concentration de substrat limit, la synchronisation et le
profil cellulaire ont t obtenus.

ACKNOWLEDGMENTS
This work was made possible through grants from the Natural Sciences and
Engineering Research Council of Canada (NSERC) and the Fonds pour la formation de
chercheurs et l'aide la recherche (FeAR).
Additional thanks are extended to the entire Falcon research group, for their
ftequent encouragement and good cheer, and to Michael Silverberg for aU his help. Of
course, Many thanks go ta my parents for their unconditionallove and for putting up with
ail my erratic moods. Extreme gratitude is also felt towards Isabelle Joubert, for her

immense support and patience throughout the duration ofthis research.

Finally, l wouId especially like to thank Dr. D.G. Cooper and Dr. AD. Rey for
their guidance, support, and fiiendship throughout the duration of my studies.

ili

TABLE OF CONTENTS

1. INftODUgION

1.1

SELF-eYCLlNG F'ERMEN'rATlON

1.2

CLASSIFICATION OF CELL POPUL.A.TION MODELS

1.3

SEOREGATED,

1
.

..

Il

SnuCJlJRED MODELS... 12

1.4 PREVIOUS MODELmO Wou

13

1.S V ALIOATION CRITERIA FOR n SCF MaDEL

15

1.6 OBJECI1VES

16

1.7 THEsIS OROANIZATION

17

CHAPnR %. mE CELL AGE MODEL

18

2.1 lNTRODuC110N

18

2.2 FOWULATION OF nIE CEU.. AOE MODEL ....... 18

2.3 A CELL AOE MoDEL FOR lNDucnON SYNCHRONY

21

2.4 SOLUTION SCHEME FOR THE CELL AOE DlSTRlBurION MODEL: MEnlOD OF CHARACTERISTICS

27

2.5

33

REsULTS AND DISCUSSION

2.6 CONCLUSIONS

38

CllA.PTER 3. CELL MASS MODEL

40

3.1 !NTRoouCI10N........

40

3.2 FORMULATION OF THE CEIJ..MAss MODEL

41

3.3

57

S0LlTl10N SCHEME

3.3.1 Galerkin Fin/te Element Method......................................................................................... 57

3.3.2 Prediclor-Coweclor Euler Scheme......................................................................................... 61

3.4 REsULTS AND DISCUSSION

63
63

3.4.1 Verification ofthe Solution

IV

3.4.2 Application a/the Ce// Mass Population Balance Madel ta the SCF Problem ......................... 75

3.4.3 Dispersion Effects within the Cell Moss Population Ba/ance Model

84

3.S MODIFICATION TO 11 CELL MAss POPULATION BALANCE MaDEL

87

3.6 REsutTS AND DISCUSSION Of mE MODIFD CELL MAss MaDEL

90

3.7 CONCLUSIONS

97

CH.APTER 4.

99

CONCLUSIONS

REnUNCES

102

LIST OF TABLES

TABLE 1. PARAMETER VALUES USED FOR THE SIMULATIONS OF 1lIE CHEMOSTAT AND
BAT MOnaS. .

64

TABLE 2. PARAMETER VALUES ESTIMATED FROM THE WORK OF WINCURE Er AL

79

vi

LIST OF FIGURES

FIGURE 1. CONCENTRATION PROFILES FORCINEI'OBACTER CALCOACEI1CUS RAG-l

OROWN usmo 'IllE SCF lEClINIQlJE

FIGURE 2. 1NTRAcYCLE CELL COUNT PROFILE FOR ONE CYCLE OF PSEUDOMONAS PUTITA
GROWN USING nm SCF TECIOOQtJE

FIGURE 3. 1NrERCYCLE PROFll.E OF DO AND CELL NUMBER. FOR ONE CYCLE OF

ACl.\'ETOBACTER CALCOAC.77cusRAG-l GROWN USINO 1HE SCF TECHNIQUE

FIGURE 4. 1NTRACYCLE DO AND CELL NUMBER PROFILES FOR ONE CYCLE OF

PSEUOO.\fO.\'AS PUT1TA GROWN USING THE SCF TECHNIQUE

FIGURE S. 1NrERCYCLE PROFILE OF DO AND CELL NUMBER OF CANDIDA UPOLYTICA

GROWN USINO nIE SCF l'ECfOOQUE

FIGURE 6. 1NTERCYCLE PROFILE OF DO AND CELL NUMBER OF CANDIDA UPOLrrICA GROWN

UsrnG nm SCF lECflNIQUE
9

FIGURE 7. 1NTERcYCLE PROFILE OF DO AND CELL NUMBER OF CANDIDA UPOLYTICA

GROWN USING nIE SCF TECHNIQUE

10

FIGURE 8. CHANGE IN DISTANCE BETWEEN 'IWO CELL UNES FOR A VARVlNG DMSION AGE .

....................................

fi

24

FIGURE 9. CELL UNES REPRESENTING nm SlEADY CYCLE SOLurION Ta THE POPULATION

BALANCE EQUATION WHEN A PERIODIC SHIFT IN nm DMSION AGE IS IMPOSED ..... 26

vii

FIGURE 10. CHARACTERISTIC CURVES SPANNINOTIIE AGE-TIME PLANE.............. 30

FIGURE Il. SIMULATION OF mE EVOLurION OF A SYNCHRONIZED CULTIJRE IN SCF

3S

FIGURE 12. 1NITIAL RECfANGULAR CELL AGE DISTRIBunON AND nIE FINAL DISTRIBtmON
OF mE SYNCHRONIZED CELL POPULATION
36

FIGURE 13. TRANsIENT PROBABILITY OF CELL DMsrON ft (m, CI) VERSUS CELL MASS M.

EFFECT OF Cs

50

FIGURE 14. TRANSIENTPROBABILITYOFCEllDMSION

EFFECT OF &

f'(m,Cs ) VERSUS CELL MASS M.

51

FIGURE 15. DISTRIBunON OF DAUOHTER. CELL MASS FOR TWO DIFFERENT g' VALUES

53

FIGURE 16. DISTRIBUTION OF DAUOHrER. CELL MASS AS ONEN BY SUBRAMANIAN ETAL.5 5

FIGURE 17. DO, LIMlTING SUBSTRATE, BIOMASS, AND CELL NUMBER PROFILES FOR A
CIMOSTAT, USINO nm INITIAL CONDmONS GIVEN BY EQUATION (77)

66

FIGURE 18. CELL MASS DISTRIBunONS FOR A CflEMOSTAT, USING mE INITIAL CONDmONS
GIVEN SY EQUATION (77)
67
u

FIOURE 19. DO, LIMITING SUBSTRATE, BIOMASS, AND CEIL NUMBER PROFILES FOR A
CHEMOSTAT, USINO 1BE INITIAL CONDmONS GIVEN DY EQUATION (78)

viii

68

FIGURE 20. CELL MASS DIS1RIBUTIONS FOR A CHEMOSTAT, USINO mE INITIAL CONDmONS
GIvm.r BY EQUATION (78)
69

FIGURE 21. DO, LIMlTING SUBSTRATE, BIOMASS, AND CELL NUMBER PROFILES FOR A
CHEMOSTAT, USING mE INITIAL CONDmONS GIVEN BY EQUATION (79)

70

FIGURE 22. CELL MASS DISTRIBUTIONS FOR A CHEMOSTAT, USING mE INITIAL CONDmONS
GIVEN BY EQUATION (79)
71

FIGURE 23. SOLUTION OF mE CELL MASS POPULATION MODEL, AS GIVEN BY nIE

EQUATIONS Il'ol REFERmeE [15]

73

FlOURE 24. RESULTS FROM nm MODIFIED POPULATION BALANCE EQUATION FOR THE DO,
LIMlTING SUBSTRATE, BIOMASS, AND CELL NUMBERPROFILES OF A BATCH REACTOR,
USINO nIE INITIAL CONDmONS ONEN BY EQUATION (77)
76

FIGURE 25. RESULTS FOR nm MODIFIED POPULATION BALANCE EQUATION FOR nm CELL
MASS DIS1'RIBunONS OF A BATCH REACTOR
77

FIGURE 26. BIOMASS, UMlTlNG SOBSTRATE, AND DO CONCENTRATION PROFILES FOR nIE
SCF SYSTEM
80

FIGURE

27. CELL NUMBER AND DO PROFILES FOR mE SCF SYSTEM....................... 81

FIGURE 28.

CELL MASS DISTRIBUllON FOR mE SCF SYSTEM

82

FIGURE 29. CELL NUMBER, BIOMASS, AND CaL MASS DISTRIBtmON PROFILES FOR A
CHEMOSTAT REACTOR, USING A NARROW INITIAL CELL MASS DIS1RIBtmON.......... 86

ix

FIGURE 30. CRrnCAL DMSION MASS Mc AS A FUNeTION OF LIMlTING SUBSmATE Cs

91

FIGURE 31. SIMULATION RESULTS OF CELL NUMBER. AS A FUNCTION OF TIME FOR mE SCF
SYSTEM, WlTII CEll MASS DMSION CON"IROL
93

FIGURE 32. SIMULATION RESULTS FOR 1HE CELL NUMBER PROFILE OF mE SCF SYSTEM
COMPARED Ta nm PROFILE OBTAINED WHENnIE DMSIONMASS IS fJELD CONSTANT.94

FIGURE 33. EFFEcr OF nIE VARYING CRITICAL DMSION MASS Mc ON nm CELL MASS
DIS'I'RIB1ITION............................................................................................. 96

Chapter 1. Introduction
1.1 Self-Cycling Fermentation
The self-cycling fermentation (SCF) process was first developed by Sheppard and
Cooper [41,42] as an improvement of continuous phasing [11,12,13].

SCF is also

described in later references [3,4,26,31,40,50,52,54]. This fermentation process is based

on monitoring a growth associated parameter while growing microorganisms in a
bioreactor. As the limiting nutrient approaches depletion, a decrease in the metabolic
activity of an organism is reflected in the measured parameter. At this time, half of the
broth is removed from the reactor (harvesting) and is replaced with fresh medium
(dosing). The action of harvesting and dosing is known as cycling, while the period
between successive dosing steps is known as a cycle.
After a few transient cycles, the system settles into a stable periodic state. Figure 1
demonstrates typical biomass, limiting substrate and dissolved oxygen (DO) concentration
profiles as a function of time for the SCF process for severa! cycles of Acinetobacter

ca/coaceticus RAG-I [53]. In this example, ethanol was the limiting nutrient. The
biomass concentration profile (top graph) is seen ta increase exponentially throughout the
cycle while the limiting substrate concentration profile (middle graph) decreases
exponentially ta a value below detectable Iimits.

The growth associated parameter

monitored during this fermentation was the DO concentration (bottom graph). Air was
supplied to the system at a constant rate. For a given cycle, the DO concentration initiaUy
decreased exponentially. As the limiting nutrient approached depletion leveI, a decrease

0.7

r----------------------------,

0.8

0.3

0.2

0.1

J..-

---J

0.5.,.----------------------------,
o..

0.3

0.1

.0.1

JL-

100 ~------------------------.....,

es
90

J_M

JI:

85

80
55
SO.f------+----+-----+-----+---~--....._--

20

eo

ao

100

120

........- - _ _ f
140

180

TIIIIC........

Figure 1. Concentration profdes for AC;lIsD6aeter clI1cDtlCSclIS RAG-11I"0wn usinl

the SCF technique [53].

in the metabolic aetivity caused the no concentration to increase. Thus a minimum in the

growth associated parameter was observed toward the end of the cycle, and once
detected, the system was allowed to cycle.
The biomass profile also demonstrates another important feature of SCF. The
biomass concentration prior to harvesting was found to be the same for all cycles. Snce
half the biomass was removed during harvesting, and was recovered by the end of the
cycle, the length of the cycle, the cycle time, must be equal to the doubling time of the
microorganism. Thus, the cells double exaetly once during cach cycle.
One advantage that this type of fermentation has over conventional batch
fermentations is that no lag phase or stationary growth phase are observed during a cycle.
These periods of slow growth are common in batch fermentations [1]. SCF aIso has the
advantage of not having long down tintes for cleaning, sterilization, etc., which are
inevitable between batch fermentations.

Thus, the microorganisms can grow at the

maximum growth rate for prolonged periods of time. Maximum growth rates can also be
achieved in chemostats.

However in these systems, the limiting substrate is ooly

completely consumed at very low dilution rates for which the growth rate is Iow [1]. SCF
has the advantage of supporting high growth rates for extended periods of time with the
complete utilization of the limiting substrate. This fermentation method has been used for
both the biodegradation of various industrial pollutants [4,26,40] and for the enhanced
production ofvarious biological products [31,41,42,52,54].
Another important feature of SCF is the synchronization of the microorganisms in
the system. Figure 2 depiets a typical cell number profile in seF for one cycle [40], while

Figures 3 and 4 show the DO profile and the cen number profile as a function of time for
two difFerent mieroorganisms [3,26]. These figures show that the total cell number in the
reactor does not increase exponentially throughout the cycles, but rather increases in a
step-like fasbion towards the end of the cycle. This synchranization in cellular division
suggests a temporal alignment of the microorganisms' cellular cycle. The location of the
$lep increase during the cycle also corresponds to where the minimum in the DO profile
occurs. This is evident in Figure 3, and in Figures S, 6 and 7 where the SCF technique
was used to grow Candida lipolytica [52]. During these runs, the fermentation was
allowed ta continue beyond the minimum in DO which corresponded to the exhaustion of
the limiting nutrient CNHthSO... The system was allowed to continue, without cycling,
until a second nutnent (glucose) was cornpletely cansumed, at which point the DO
concentration was seen ta rise rapidly. The system was only allowed to cycle upon this
sharp increase in DO.

AlIowing the cycle to continue after CNHt)2S0.. had being

exhausted was termed extended nutrient starvation. Cen synchrony was still rnaintained
using tbis mode of cycling, with the step increase in cell number corresponding to the
depletion of the limiting nutrient. No other data exists on the cell number profile during
extended nutnent starvation using SCF. However data obtained by Dawson [11,12,13])
doing work on the synchronization of organisms using continuous phasing, showed that
cellular division also occurred upon exhaustion ofthe limiting substrate.
The ability of this method to generate and maintain synchronized cell populations
is very useful for the study of cell cycle events.

The synchronization of the

microorganisms tan result in an amplification ofcellular events. Since a large fraction of

2.2 ~--------------------------.,

End of Cycle

1.2

1-+-------+------+----+------+------+---~

10

eo

20

Figure 2. Intracyde ceU count prorale for one cyde of Pse"domolltB p"tittlgrown
usinl the SCF tecbnique [40].

..

to

End of Cycle

10

70

ISO

J~
J~

50

l
ot

40

30

20
tO

:u

1
~

a
ri
.a

2.3

2.t

1.1 ~

t.7

1.5

1.3

t.t

E
:1

o.a ,

0.7

0.5
0

tO

20

30

.
40

eo

10

70

10

90

n.'(.IIIUI.'

Figure 3. Intercycle prorlle of DO ADd ceU Dumber for one cycle of Acinetobacter

calcoaceticlIs RAG-l growa UliDI the SCF technique [3].

SlO

ao
70

80

JI

50

~J
l

40

~ 30
2D
10
0

1.5

_ 1.4
E

~ t.2

1
~

1.t

1
~

0.9

o.a ,

0.7

0.11

1.3

10

20

.
30

40

50

10

70

10

ThIl. (............

Figure 4. Intracyde DO and ceU number profila for one cycle of PselldolllOftllS
plltita grown usinl the SCF technique [26].

8 5 . - r - - - - - - - - - - - - - - -..........

80

65
60
18

__J

~-----------------.,

17

16..

15
;:; 14 .
u

c 13 ...

i-- 12..
5

11

u
1i 10
u 9

8..10.----------------'
o
100
400
300
200
nme(mlnutal

Figure 5. Intercyde prorde oC DO and ceU Dumber oC c.lldidtJ lipolytica growD UliDI
the SCF technique [52).

100

SIS
c

80

01

15

1i:~

la
-1

1~ ea

75

70
20

U
If

..

1.

12

1e

18

10
Il

100

200

300

400

!lOO

eoo

.
700

TI... Cftlln.....

Figure 6. Intercycle profde of DO and ceU number of Candida lipolytictllrown usinl

the SCF technique [52].

95
90
c

11
JI

80

j f!

75

85

70
13

12

11

10

1
.,a
.-

1
U

"

7
8

.
100

200

300

..

.00

500

Tlm. (1IIlnutal

Figure 7. Intercyde profile of DO and eeU Bumber of Calldida lipolyticlJ grown usinl
the SCF technique [52].

10

the cell population go through cell cycle events simultaneously, the entice system can be
studied as a representation of single cell aetivity. The faet that the SCF cycles are very
repeatable, and that the duration of these cycles correspond exactly ta the
micro 0 rganism's doubling time, aIso facilitate these types ofstudies.
1.2 Classification of Cell Population Modela

8iological systems, and their interactions with their environment, are very complex
and the models used ta study them generally do not attempt to capture all minor details.
For tms reason, scientists and engineers have developed models that usually deal with
specifie and fundamental aspects of biological systems. Engineers have derived a host of
mathematieal models with the objective of controlling and optimizing biological processes.
This section deals with the classification ofthese classical models as proposed by Tsuchiya

et al. [45] and discussed by Ramkrishna [36,37] and Balley and Dllis [1].
A mathematical model of a biological system can be c1assified as segregated or
non-segregated. Segregated models recognize the faet that a population is composed of
distinct individuals. Non-segregated (distributed) microbial models, such as Monod's
models [1], do not recognize individuals ceUs but lump them into an average biophase
such as dry biomass concentration.
struetured or non-struetured.

Microbial models can also be charaeterized as

Struetured models take into account the state of the

microorganisms. In the case of segregated struetured models, the population is treated as

individual cells which can be dift"erentiated from one another. This is accomplished by
specifying the state of the microorganism. For example the chemical composition of the
ceU, the cell age, the eeU mass, the morphology or ceU size, or a combination of indices

Il

can he used to indicate the physiologica1 state of the microorganism. In the case of a
distributed model this would imply the specification of the state of the lumped biophase.
Non-structured models cannot differentiate between individual ceUs, or between different
states of the lumped biophase. Finally, microbial models can be classified as detenninistic
or stochastic.

Although cellular division and birth processes are thought ta be

probabilistic [45], for large cell populations, these processes can be readily described in
terms ofdeterministic funetions [1,36].
The population balance models dealt with in tbis study are segregated, structured
microbial models.
1.3 Segregated. Structured Modela

Segregated, struetured microbial models treat cellular populations as distinct

individuals which can be differentiated trom one another. This differentiation between
organisms can be charaeterized by a number of different indices of physiological state.
Ramkrishna [36,37] and Fredrickson et al. [18] bath discuss the mathematica1 framework
for a general population balance model. They discuss the case when an arbitrary number
of variables are used ta describe the state of the organisms, and the state of the growth
environment. However, from an experimental point ofview, monitoring a large number of
physiological indices at once can praye ta be impraetical.
Rey and Mackey [8,38,39] have worked with a population balance model where
age and cell maturation were considered. The proliferation stage was considered to be
composed of four distinct major phases (Go, GI , S and Ch). This description of cellular
proliferation lead ta the formulation of a differential, delay equation. Rey and Mackey

12

studied the rich dynamic behavior exhibited by the equation. This model of cell replieation
is weil suited for the eucaryotic cell cycle which can be described in terms of discrete,
sequential events.

In the case of the microbial cell cycle, there is much less differentiation between
the various parts of the cycle. For example DNA synthesis can oecur throughout most of
the cycle and proceeds in parallel with other growth processes such as protein and RNA
synthesis [1]. This is in contrast with the eucaryotic ceU cycle were DNA synthesis occurs
only in the S phase ofthe cycle.
Microbial cultures are often mode1ed using a single index of physiological state. In
this worle, a cell age model and a ceU mass model of SCF will be developed and used.

1.4 Previous Modeling Work

The proposed population balance models presented in this thesis are not the tirst
model of SCF to be developed. Wmcure et al. [53] bas developed and solved a nonsegregated model of the system. The constitutive equations used in tms Madel were the
Monod constitutive equations which were modified to account for the instantaneous
cycling ofthe system. The model predieted the behavior ofthe biomass concentration, the
limiting substrate concentration, and the DO concentration. The equations were:

dX
= (PIUXC.)X_
~jX6(t-t.
dt
C +k
~

J-l

.)

(1)

lIuaOt,)

(2)

13

where X = biomass concentration (gIL),

CI, Co =lirniting substrate, and DO concentrations (gIL),

CSF, CaF =limiting substrate, and DO concentrations ofthe fresh medium (gIL),

Ys, f o
-x -x
t

= limiting substrate, and DO yield coefficients,

=time (br),

J.lmax = maximum specifie growth rate (br-I),

le. = saturation constant (gIL),

kLa = liquid side mass transfer coefficient (he- I ),

Co = saturation concentration of dissolved oxygen in the medium,

f =emptyinglfill fraction,

S(t) = delta Dirac function,

tmin 02J

=time at the DO minimum (br),

= cycle number.

These differential equations, along with the appropriate irtial conditions, were integrated
numerically.
The model was able to prediet the major macroscopie features of SeF. It captured

the stable periodicity of the system, the biomass production, the limiting substrate
consumption, and the DO concentration profiles. When comparing these results with

14

experimental data, the simulation results are seen ta capture the major trends along with
the end of cycle values. However, the model output was seen to become out of phase
when compared to experimental data.

Wmcure attnouted this to the faet that

instantaneous cycling was assumed in the model construction. This explanation seems
unlikely since taking account of the finite cycling time would prolong the cycles of the
model. The experimental data suggests that the simulation cycles were longer than those
of the actual data. A possible explanation could be that the kinetic parameters of the
system were poorly estimated or might have changed

wi~

time due ta adaptation of the

organisms to the growth conditions of the system. In faet, a1though the predicted end-ofcycle values correspond ta that of the experimentai values, the simulated values took
longer ta reach these end-of-cycle values.
The model was also able to predict the stability of the system when the
emptying/filling fraction was other than 1/2. However, this model does not reveal any
information on the total cell number profile nor does it provide any insight iota the
l

synchronization ofthe organisms. To study the phenomenon of cell synchrony a different

model had to be developed.
1.5 Validation Criteria for the SCF Mode'

Any new model of SCF should be able to capture at least the main macroscopie
features accounted for by the previous model. In addition, the cell number profile and the
feature ofsynchrony should be explained by tbis model. OveraU, the experimental features
to be captured by the proposed new model were:

The stable periodicity ofthe system, including the cycle length

IS

The macroscopie profiles (biomass, limiting substrate and DO concentration)

The total cell number profile and eell synchrony

1.6 Objective.
This work deals with the modeling and simulation of the SCF process. The
approach taken is to develop and solve a segregated, structured model ta study the
system. Two different models were considered: a cell age model and a cell mass model.
Bath these models have been used to study various microbial systems, and can give rise ta
very different cell number profiles. The specifie objectives ofthis study were:

1. Develop cell age and cell mass microbial population balance models for SCF.
2. Develop numerical methods and algorithms to solve the population balance models for
SCf.

3. Select the MOst appropriate population model using criteria based on available
experimental data.
4. Validate the model and select the model parameters using available data.
S. Provide a fundamental understanding of the various physieal and biological processes

operating in the SCF process.

6. Establish the process conditions and mechanism that May lead to the convergence of
cell synchrony.

16

1.7 Thesis Organization

This thesis is organized into four chapters. Two different population balance
models were applied ta simulate the SeF process. Each of these models is dealt with in
separate chapters. Chapter 2 deals with the development, application, and discussion of
the cell age population balance applied ta SCF. Chapter 3 discusses the development,
application, and discussion of the cell mass population balance applied to SCF. In both
these chapters the simulation results are compared with experimental data in order ta
determine their suitability to model the SCF process. Chapter 4 is an overall summary of
the work. Finally, an Appendix is also included, and contains the computer program that
was written to numerically solve the cell mass model for different fennentation systems.

17

~ter 2.

The Cell Age Madel

2.1 Introduction

This chapter deals with the fonnulation of a segregated, structured microbial

population balance model for the SCF process, which used ceU age as the single index of
physiological state.

eeu age distribution models have the advantage that no assumptions

have ta be made about the single eell growth kinetics. In addition, the population balance
equation is simpler for the age distribution when compared ta other types of distribution
models. However cell age cannat be measured experimentally unless a cell has been
followed since binh, and therefore its predictions can not be validated without further
assumptions.
The derivation that fol1ows is based on work by Trocco [46] and the resultant
population balance equation is known as the Von Foerster equation. The cell age model is
also discussed in [45].
2.2 Formulation of the Cell Age Model

Given a cell population, let N.(t) be the number of ceUs, al time 1, that have ages
between a and a + a. Assuming that lim.-.o[AN.(t)! a] exist, we can define the cell age
density funetion o(1,a) = lima...oN.(t)! l. Integrating 0(1,1) over all ages (a) gives:

GD

N(t)

=fn(t,a)da

(4)

18

where N(t) is the total cell number in the culture (cells/volume). Therefore, the number of
eells which are in the age interval (a, a + Aa) at time t is equal to o(1,a) a. In a small time
interval dt, the age of each cell increases by dt. It is worth ooting here that the units of

time and eell age should be the same and that cell age can ooly be positive. In addition, a
eell of age zero is defined as a eell that was just ereated from cellular division. For the
time interval dt, the following expression can be written:

net + dt, a + dt) Aa + cell death = n(1,a) Aa.

(5)

Cell death is assumed to he proportional to the number of cells in a given cell age group
n(t,a) a, and to dt. It can be written as:

ce// death = -.(t,a, ...)n(t,a)&ldt

(6)

where is the loss function (l/time) and could depend on 1, a, and other parameters of the
system. Equation (5) can he rewritten as:

n(t + dt, a + dt)

~a =

n(t,a) ~a - (t,a,... )n(t,a) Aadt.

19

(7)

Expanding net + dt, a + dt) in powers of dt yields:

Il

net + dt, a + dt) = n(t, a) + -dt + -dt+...O(dt 2 ).

~
a

(8)

Dividing equation (7) by a and substituting in equation (8) gives:

on(t,a)
l

-~~+

n(t,a)
= -l(t,a, ... )n(t,a)
oa

(9)

which is the Von Foerster equation [46].

For the integral in equation (4) to converge, n(1,8) as 8

~ co

must go to zero.

Integrating the Von Foerster equation trom a = 0 to a = CI) results in the total cell balance:

dN(t)
d
= n(t,O)t

Jco

l(t,a, ... )n(t,a)da.

(10)

To solve equation (9) the boundary and initial conditions must be specified. The
boundary condition is expressed for a = 0 as:

co

n(t,O)

= 2Ir(a)n(t,a)da

(11)

where r(a) is the division modulus defined 50ch that the probability that a cell with age 8

20

will divide between t and t + dt is equal to r(a)dt. Equation (11) is the renewal equation

which accounts for the number of newbom ce1Is as a funetion of time. Here, binary
division is assumed.
Division is expressed in terrns of a probability funetion which bas to be integrated
over all ages.

For simplification, and without lost of generality for the upcoming

discussion, it will he assumed that all cells divide at the sarne age e. The renewal equation
can then be rewritten without the division modulus and the integral as:

n(t,O) = 20(t, S).

(12)

The initial condition for the population balance equation is the initial age
distribution:

n(O,a) = Ilo(a).

(13)

2.3 A CeU Age Model for Induction Synchrony

Two different methods are generally used to obtain il synchrony in pure cultures,

selection techniques and induction techniques [5,12,33]. Selection techniques usually

involve the physical isolation of ceUs that are close together with respect ta their
progression through the cell cycle. These ceUs are often differentiated trom the rest ofthe
population based on marphological differences. For example, centrifugation is often

21

employed to segregate cells of ditrerent size and mass in a gradient. Cens having similar
charaeteristics can then be isolated and used as an inoculum into a sterile medium. These
cens will generally produce a synchronous culture which can exhtbit sorne cenular
synchrony for a few generations. The synchrony is eventually lost due to randomizing
factors.
Induction techniques usually involve imposing sorne shift in the growth
environment of the organisms to bring about cen synchrony. This can be accomplished
through single..shock treatments were a single disturbance is introduced which causes the
cells to align themselves with respect to their cell cycle, or through periodic shocks where
a disturbance is applied to the system at fixed time intervals. This later method has the
advantage of providing an environmental pressure to rnaintain cell synchrony for
prolonged periods oftime.
Hjortso has proposed a cell age model for induction synchrony [23].

Cell

synchrony was studied using a cell age distribution model in which the age at division,
was subjected to periodic forcing.
distribution assuming no cell death,

n(t, a) + l1(t, a)
t
a

e,

The population balance model for the cell age

=0, cao be written as:

=0

(14)

with the renewal equation

0(1,0) =20(1,0).

(15)

22

Under certain conditions, periodic shifting in the age at division resulted in

synchronized il populations. This change in division age could result ftam a change in
the growth environment.

This idea was exploited by Hjortso and Nielsen [25] who

modeled oscillations and partial synchrony in continuous cultures of Saccharomyces

cerevisiae, using an age distribution model. They reasoned that, as the limiting substrate
concentration increased, the duration ofthe cell cycle length should decrease.
Figure 8 shows the growth and division of ceUs along two ditrerent cell lines. Cell
lines represent the growth curve, in the age-time plane, of ceUs having the same cell age.
The graph depicts the behavior of two celllines before and after division when the age at
division, e(t), decreases with time. The age difference between these two ceIllines prier
to division is .180, while their difference after division is Aal_ Since the cell lines have a
slope of 1, the age difference between two given cell lines is equal to the distance
separating them in time, At. Assuming binary fission, as At, becomes differentially smalt,
the following number balance over dividing ceUs can be written:

n(t,O)dtl =2n(t,0)dte,

(16)

A relationship between the two time intervals can be expressed as:

23

CellAge

Time

Figure 8. Change in distance between two ceillines for a varying division age 8
[23].

24

where 480 is the change in the division age during the time interval

4th

and is positive

when 0 increases over Atl. Thus, it can be observed that if the division oftwo celllines
occurs while the division age is decreasing (A80 <0), the distance in time between them
will be smaller after they have both divided. Therefore the cells growing along these cell
lines will be closer to each other in the age domain, after division.
As to becomes differentially small, the following can be written:

dia

=1- d0 ,

(17)

dl

dt)

and the renewal equation becomes:

n(t,O) =

(1- ~)2n(t,e).

(18)

By imposing a periodic shift to the division age, Hjortso was able to simulate the
induction of synchrony. Figure 9 depiets the steady state behavior of cell lines when such
a trend is imposed ta the division age. Hjortso reasoned that the condition required ta
ensure the induction ofsynchrony was:

(19)

25

Cell Age

t ..
/

/
/
/
/

/
/

Time
Figure 9. Ceillines representiDI the steady cyde lolution to the population balance
equation when a periodie sbift in tbe division age il impoled. The solid lines
represents the stable .ttraeton while the dashed lines represent the unstable
repellen.

26

where

't

is the period of 8(t), and al and a2 are the lower and upper mits of 8(t),

respectively. Assuming that 0(t) bas no local extrema between al and a2, and that the
condition represented by equation (19) is fulfilled, then for each cycle of0(t) there will be
two celllines which divide when 0(t) = 't. One ofthese celllines will interseet the division
age when 0(t) is increasing and one will intersect when 0(t) is decreasing. These cell
lines represent the steady state cycle solutions to the population balance equation. The
cells in these cell lines will always divide at the same relative position in each of the
dividing age cycles.

Hjortso abserved that amang these cell lines, the ones which

divided when 8(t) was decreasing were attracting neighboring cell lines, while those
which divided when e(t) was increasing repeUed their neighboring celllines. These cell
Unes were tenned attraetors and repellers, respeetively.

The cell Hnes between two

repellers will therefore converge onto the attraetor cell line in this region.
Hjortso also demonstrated that a rich array of dynamic behavior could he achieved
when the periadic forcing did not meet condition (19). He described examples where
bifurcations gave rise ta muitimodai synchrony, and he discussed cases exhibiting behavior
similar to period doubling, halving, and chaos. A brief discussion of how 0(t) could be
modeled was also given.
2.4

Solution Scheme for the Ce" Age Distribution Model: Method of

Characteristics

An analyticai solution of the cell age distnoution model can be obtained using the

method of charaeteristics [24]. In this method, partial differential equations are changed

27

into sets of ordinary differential equations (ODEts). These ODEts are then solved along
characteristics curves in the plane spanned by the two independent variables.

The population balance equation to be solved was:

n(t, a) + n(t, a) = 0
t
a

(20)

with boundary condition:

(21)

and initial condition:

n(O,a) = l1o(a).

(22)

Equation (20) can be written as an ODE such that:

dn(t, a)
dt

= n(t, a) +
dt

da n(t, a) = 0
dt
a

28

(23)

where the cell age growth rate, da = l, is the differential fonn ofthe charaeteristic curves,
dt
which can be integrated to give:

a=t+e

(24)

where 8 is a parameter. Figure 10 shows the family of charaeteristic curves (straight Unes)
which span the age-time plane, along which equation (23) cm be directly integrated. For
&

> 0, equation (23) is integrated from the initial condition described by equation (22),

over the independent variable time, while for &<0, the ODE can be integrated ftom the
boundary condition equation (21), over the independent variable cell age (see Figure 10).
For &<0, the ODE to be integrated is:

dn(t,a)
da

where dt
da

dt n (t ,a) + n (t, a)
da
t
a

(25)

=1 is the same characteristic curve as equation (19).

For & > 0, a> t, substituting equation (19) and integrating equation (23) over time
yields:

II(t.t+~)

fdn=fo
..(0.&)

fora>t

29

(26)

CellAge

8>0

8<0

(/)

=
o
......
......
~

'"0

=
o

........

~
......
......
....c
~

Boundary Conditions

Time

Figure 10. Characteristic cunes spanning the aae-time plane. For E < 0, the spatial
ODE is inttlrated usinl the bODodary condition (21), and where E ~ 0, the tilDe
ODE is integrated usinl the initial condition (22).

30

and

n(t,t +e) = n(O,e)

for a > t.

(27)

for a > 1,

(28)

Substituting for & (24),

n(t, a) = n(O, a - 1)

where n(O,a-t) is the initial condition l1o(a-t). For & < 0, a < t, substituting equation (19)
and integrating equation (25) over cell age gives:

11(11-".11)

Il

11(-&.0)

fdn=fo

for a <1,

(29)

for a < t.

(30)

for a <1,

(31)

and

n(a - s, a) = n(-s,O)

Substituting for E,

n(t,a) =n(t -a,O)

31

where n(t-a,O) is the boundary condition n(1 - a,O) =

(1- ':) 2n(1 - a, e). Therefore the

solution ta equation (20), with boundary and initial conditions described by equations (21)
and (22), is:

n(t, a)

="0 (a - I)

for a>t,

(32)

for a<1.

(33)

and

(1- :)211(1 -a,e)

n(t, a) =

The previous model refers to an isolated cell cycle.

Next we discuss the

application orthe cell age model to the SCF process. In the case of SCF, equation (33) is
simply solved for each cycle. As discussed in the introduction, upon cycling, half the cell
population are removed trom the bioreaetor. In this model, this is accomplished by
multiplyjng the cell age distribution at that tinte by a factor of .!... This new distribution
2
will become the new initial distribution, and equation (33) will be solved for the new cycle.

The independent variable t in equation (33) should therefore he reset to zero after cycling.
The overall fermentation time can he obtained by adding the successive cycle times.

32

2.5 Resulta and Discussion

The induction mechanism proposed by Hjortso [23] depends on the relationship

between the substrate concentration and the Mean division age.

As the substrate

concentration reaches limiting concentrations, the growth rate of the microorganisms

decrease. This is reflected in the model by an increase in the doubling time of the
microorganism through an increase in division age.
As observed in Figure 1 and in other studies [3,4,26,31,41,42,52,53,54] the

limiting substrate concentration decreases monotonically during a cycle. This decrease in

substrate concentration eventually leads to a decrease in the growth rate of the
microorganisms which causes an inflection point in the DO trace.

Eventually the

metabolic aetivity reaches such a low level of aetivity that a minimum occurs in the DO
profile. This eventual decrease in growth rate is retleeted, in the age model, by an increase
in the division age, since the single cell growth rate, da, remains constant and equal to 1.
dt

A schematic diagram of the behavior of the division age in SCF was shawn in
Figure 9 of section 2.3. The division age is shawn ta increase manotonically throughout
the cycle up to the detection of the minimum in DO at which point, half of the reador
volume is harvested.

Upon dosing, the rapid increase in the limiting substrate

concentration would cause a sudden decrease in the division age. Therefore, in each
cycle, the division age goes through exaetly one period, with a monotonie increase and
decrease between the two cell age values al, and a2.
In section 3.2 it was aIso proposed that the condition for induction synchrony (19)

was that

QI

S 'r S tlz where t is equal to the cycle length. In SCF,

33

in order ta obtain

constant end-of-cycle biomass and cell number, !bis condition has ta be met eventually. If
t

were ta he less than al, the organisms would not all have time to divide during each

cycle, and the end-of-cycle biomass would decrease. If

were greater than

82,

microorganisms would have gone through more than one full cell cycle during each

the

SeF

cycle, which would lead ta an increase in the total end-of-cycle biomass concentration and
cell number tram one cycle ta another. The faet that, once the system reaches a stable
periodic state, the amount of end-of..cycle biomass and the end-of-cycle total cell number
remains constant for each cycle, implies that equation (19) is satisfied.
Under this condition, Hjortso argued the existence of steady-state cell lines. The
steady-state cell line which interseets the decrease in division age was described as an
attraetor celllines. AlI other cell line in the vicinity of this ceU line will be attracted ta it,
independently of the initial cell age distribution
model, induction syochrony will occur in

1.

Theretbre, according to the cell age

seF through the convergence of cells onto the

attraetor ceU line.

The sudden decrease in division age occurs during the dosing stage of the process.
Once cell synchrony has been achieved, the ceU age model prediets cellular division during
the dosing stage. Figure Il depicts the resulting cell number profile as a funetion oftime.

If the initial distnbution was such that all ceUs layon the repelIer llline, the periodic forcing of the

division age would not aff'ect the overall il age distnoution. However in a real system, random etrec:ts in
the division cycle would cause ceUs to faIl away from the repeller cell line. These ceUs would then travel
towards the stable attraetors.

34

......

1.8 , - - - - - - - - - - - - - - - - - - - - - - - ,

~ 1.6

1.4

; ' 1.2
o
... 1.0
)(

t
.CI
E

0.8

0.4

:s
Z

0.6
0.2
0.0

-+-----+-----+----+----+-----+-----1

100

200

300

400

500

600

Tlme (hours)
Figure Il. SimulatioD of tbe evolutioD of. syncbronized culture in SCF for No =
109 cells, al = 0.75 br, a2 = 1.25 br. The pattern evolveel shows that doubling in cell
Bumber occun immediately after cydiDI. This profde reluits because of condition
(19).

3S

2
.!!
"i

--

(J
"r
C

)(
~

E
:J

z
li

2.0 ...
1.8 ...
1.6 .~
1.4 .~
1.2 .~
1.0 .~
0.8 .~
0.6 .~
0.4 .~
0.2 .
0.0

1.2

li

1.0

-.
....
.z
u
0

)(

10

20

30

40

50

1.4 -

.!!
==

0.8
0.6

0.4

-15z

0.2

:J

(J

0.0
0

60

Cen Age (hou,.)

Figure 11. Initial reetangular ceU ale distribution (top), and the final distribution of
the syuchronized ceU population (bottom).

36

Figure 12 shows the initial cell age distnDution and final cell age distnoutions irnrnediately
following a dosing stage ofthe fermentation.
From Figure Il, it can be seen that the SCF ceU number profile predieted by the
age model is qualitatively different from the profile observed in experiments (figures 2 to
7). The model does prediet ceU synchrony, however the step increase in cell number

occurs at the beginning of the cycle. Experimental evidence suggests that cellular division
occurs near the end of the SCF cycles. The cell age model can not capture the qualitative
eell number trend which is seen in experiments.
In Figure Il, the cell number profile is seen to inerease linearly for the initial
cycles. For an initial reetangular cell age distnoution and a constant division age, the cell
age population balance model prediets a Iinear increase in cell number for eaeh doubling
period ofthe reetangular distribution. That is ta say, for an initial cell density No, the total
cell number increases linearly fram No to 2No with a slope of No , where T is the doubling
T
time of the organisms. The total ceU profile will then increase from 2No to 4No in a linear
fashion, this time with a slope of 2N
T

The next doubling period will have a slope of

Therefore, a1though each doubling of the population number oceurs in a linear

fashion, on a larger time scale, the total eell number increases exponentially 50ch that:

(34)

37

where N is the total eell number and t is the fermentation time after inoculation.
The actual division age profile most likely would not foUow a linear inerease as
shown in the Figure 9. This assumption of a linear increase does not lint the argument
which was made above.

The induction of cell synchrony and the

~ocation

of the

simultaneous division within the cycle will be the same as discussed above as long as the
inerease in division age contains no local extrema. Similarly, the decrease in the division
age brought upon by the rapid addition of substrate' ta the reactor during dosing was
shawn in Figure 9 ta be an instantaneous step decrease. A1though there probably is a time
delay associated with the response of the division age ta the change in the growth
environment, !his time delay is probably quite small as seen in the DO trace after dosing.
The addition of fresh medium to the reaetor quickly causes the DO concentration to
resume its decline saon after cycling. Again the assumption of a step decrease in division
age does not limit the argument which was made.
2.6 Conclusions

The cell age population balance equation, along with the induction mechanism for
cell synchrony proposed by Hjortso [23] was used ta simulate the cell number profile of
the SCF process. This equation was solved usmg the method of charaeteristics. Although
this model predicted cell synchrony when cycling was imposed on the system, the resulting
cell number profile was not comparable with the experimental cell number profile. The
predieted ceU trend was in faet opposite to the experimental trends. The simulations gave
a ceU number profile which increased suddenly at the beginning of each cycle. The cell
number profile observed in the laboratory bas the ceU number remaining mostly constant

38

at the beginning of each cycle, with a sudden step..lke increase towards the end of each
cycle.

This comparison with experimental data suggests that a il division model

govemed strictly by cell age is not adequate to simulate a cell culture where the growth
rate is not constant.

39

Qb!pter 3. Cell Mass Model

3.1 Introduction

A segregated, structured population balance equation using a single index of

physiological state, cell mass, was developed by Eakman et al. [15]. They used this
equation to model rad shaped and spherical single ceU microorganisms, growing in
continuous and batch cultures. The steady-state solution was also found for both types of
microorganisms for the eontinuous culture. A derivation is also given in [22]. The
derivation given in the next section follows the method ofEakman et al. [15].
Sorne advantages of ceU mass models over cell age models include the fact that cell
mass can be more easily correlated to system variables such as biomass concentrations,
substrate concentration, and DO concentration. Also the mass of the cell can be related to
cell volume or eeU length whieh ean be measured directly at one instant in time whereas
cell age can only be detennined by observing a panieular eeU for a large period of time.
Finally, statistical quantities distnouted on eeU mass as an index of physiological state
show a smaller variance than the statistical quantities distributed on cell age. The work of
Hannsgen and Tyson [20,21,48,49] studied the steady-state cell size distributions in
probabilistic models of the cell division cycle and found that the age at division is roughly
twice as variable as size at division. Also it wu found that the average size of eells in a
growing culture increases approximately exponentially with specifie growth rate.

In

addition, it was shown that for exponential growth, models that use eell age as a single
index of physiologieal state cannat generate stable eell-size distributions.

40

3.2 Formulation of the CeU Ma Model

The number-mass balance on live cells of mass m ta m + dm avec a time interval t
ta t + dt in a continuous reactor can be expressed as follows:

[CeUs growing into the interval m to m + dm] - [Cells growing out of the interval m ta m

+ dm] - [CeUs trom the interval m to m + dm leaving the reactor] - [Cells in the interval m
to m + dm dividing] - [Cells in the interval m to m + dm dying] + (Daughter cells bom into
the interval m ta m

+ dm from cell division] = [Change in the number of cells in the

interval m ta m + dm during the time interval t to t + dt]

(3 S).

CeUs that passed the eeU mass m during the time interval t to t + dt have a eeU
mass between m - dm to m at time 1. If we define r(m, c.> as the rate of mass increase of
a ceU of mass m in an environment with substrate concentration C. [gIL], we cm write
dm = r(m,Cc)dt. It is assumed that a vessel is perfectIy mixed and has a sterile feecl and
an eftluent stream leaving the reactor with the same composition as the contents of the
vessel. Assuming also a constant culture volume V (L], a volumetric flow rate Q for both
the feed and effiuent streams [IJhr], and that the ceUs replicate through binary fission, then
providing that dt and dm are very small, the number-mass balance can be expressed as:

41

VN(t)f(t,a)r(a,Csk)dt - VN(t)f(t,b)r(b,Csk)dt

-QI N(t)f(t,m)dmdt
a

Il

-vI N(t)f(t,m)r'(m,C6 )dmdt -vI N(t)f(t,m}8(m,C6 )dmdt

a

(36)

a
Il

+2vI
a

b
l
Jrl(m ,Cz)N(t)f(t,m')p(m,m')dm'dmdt = -IVN(t)f(t,m)dmdt
CI)

aa

1ft

where each term in equation (36) correspond to that in equation(35), and N(t) is the total
population density in the vessel at time t [cellsIL], a and b are the lower and upper limits,
respectively, of the cell mass interval m to m + dm [g], CIk is the concentration of the klh
substrate in the reactor [g/L], and

f(t,m)dm

= fraction

r' (m, Clit ) dt

p(m,ml)dm

= fraction ofdaughter cells ofmass m to m + dm obtained from a cell of

of cells at time t having a cell mass of m to m + dm, g-l

fraction of cells ofmass m at lime t that divide in time t ta t + dt, br- l

mass m' al fission, g-I

Sem, C.) dl

= fraction ofcells ofmass m at time t that die in lime t to t + dt, br- l .

The second to last term in equation (36) represents the cells which enter the mass interval
a ta b through cell division and the 2 appears because binary fission is assumed.
The first two terms in equation (36) correspond to the difference between the cells
which grow into the mass interval a to b and the ceUs which grow out of this nterval.
This expression is the net change in the total number of cells that have a cell mass in the
interval a to b during the interval t to t + dt, and can also be written as:

42

VN(t)f(t,a)r(a, C. )dt - VN(t)f(t,b)r(b, Cst)dt

-vi N(t)-[f(t,m)r(m,C.)]dmdt
an

(37)

(1

Substituting Equation (37) into (36) and rearranging, one can obtain:

H-W~,m)
-G

[r(m,C~w(t,m)] + 21r'(m' ,C.)W(t,m')p(m,m')dm'

(38)

+ r'(m,C ) + 0(m, C ) W(t,m) }m = 0

where W(t,m)

= N(t)f(t,m), and e = V/Q.

Since this expression holds for arbitrary mass

intervaJs a to b, the integrand must be equal to zero everywhere. Therefore:

JW(t,m) + [r(m,C'k)W(t,m)] = 2jr'(m',C'k)W(t,m')p(m,m')dm'

t
m

-(~ + r'(m,C'k) + 0(m,C. )W(t,m)

(39).

Equation (39) is the partial-intep difrerential equation of the ceU mus model. The

dependent variable is W, and the two independent variables are lime (t) and cell mass (m).
In arder to obtain the boundary condition for the cell mass modeL an independent

43

balance on the total eell density N(t) must first be eonsidered. This balance is expressed as:

dN(t)
dt

=2jr'(m,C.)W(t,m)cbn- N(t) 0

je(m,C.)W(/,m)dm

(40).

By integrating the eell mass model, equation (39), over aIl possible values of

nt,

one

should recover the same cell density balance. By comparing these two expressions, the
boundary condition on equation (39) can be obtained. From the definition off{t,m) which

is the fraction of ceUs at time t with cell mass ofm to m + dm, we find that:

f f(/,m)dm = 1
II

(41)

since aIl cells have a positive mass, and therefore:

II

N(t) = IW(t,m)dm.

(42)

With these results equation (39) can be integrated to become:

44

dN(t)

dt

21 [r'(m',C.)W(t,m')p(m,m')dm'dm

+r(oo,C.)W(t,ao) -r(O,C.)W(t,O) =

coco

a.

N(t)

co

(43)

co

--(}-- Ir'(m,C.)W(t,m)dm- 18(m,C.)W(t,m)dm

o

Daughter cells barn fram cell division can only have a mass between zero and that of the
parent cell, therefore:

m'

I p(m,m')dm = 1

(44)

If we interchange the order of integration of the first term on the right band side of
equatian (43), we abtain:

coco

2f ff'(m' ,C.)W(t,m')p(m,m')dm'dm =2Jf'(m' ,C.)W(/,m'~'

Om

(45)

Substituting equation (45) into equation (43) gives:

dN(t)

co

- - + r(oo,C.)W(t, (0) -r(O,C.)W(t,O) = 2Ir'(m' ,C.)W(t,m')dm

N(t)

-8- f8(m,C.)W(t,m)dm
co

4S

(46)

Since equation (46) and equation (40) must be the same, this requires that:

(47)

and since cells can only arise trom pre-existing cens and no cells can grow from mass zero,

r(O, C.) =0

(48)

Thus, the boundary conditions on the solution ofequation (39) is:

r(CX),C.)W(t,cc) = 0

(49)

which says that the convective tlux vanishes as m ~ cx).

Eakman et al. [1 S] expressed the rate of mass increase of a single cell as the
difFerence oftwo terms:

rem, C * ) = r' (m, C .) - r' (m)

(50)

where r' (m, C lit) is the rate of mass increase and r' 1 (m) is the rate of mass released by
the cell. Eakman et al. [15] postulated that since any mass uptake by the cell must be
done through the cell's surface, the rate of mass increase should be proponional ta the
surface area of the celL They a1so assumed that the mass released ftom a il was most

46

likely the product of catabolie reaetions and that the rates of these reaetions are
proportiona! ta the ceU mUI. The eeU growth rate can thereCore be written as:

rem, Ca) = 0(C IIc)S -

~m

(51)

where 0(C Ile) is the mass flux aeross the eell surface [gI(cm2hr- I)], S is the cell's surface
area [cm2

],

and J.lc is the specifie mass release rate [br-Il. Although the mass model is

derived for any number of substrates, it will be assumed later that the kineties of cell mass
growth rate is a function of a single limiting substrate Ca. This approach was used by
Eakman et al. [15]. The rate of mass increase r(m,c,) can therefore be defined as a

function of cell mass and of a single limiting substrate C.. The mass flux across the cell
surface 0(C .) is described by Michaelis-Menten kinetics which could refleet active
transport acress the cell's surface, Of could result trom Fickian diftsion of substrate
across the membrane. The specific mass release rate J.1c is assumed ta be constant.
Although these kinetic expressions were used by Eakman et al. [15] and are used here in
this study, the expression for the cell growth rate r(m, CI) is not restricted to Michaelis..
Menten behaviof, nor is it necessarily restrieted to tirst order dynamics.

The rate

expression could be adapted to any kinetie behavior observed in a system. The mass flux
across the cell surface 0(C .) is defined as:

(52)

47

where J.1' is the maximum mass flux [gI(cm2hr)] and K. is the saturation constant [gIL].
The seometry ofa coccus is assumed to be that of a perfeet sphere, while that ofa
bacillus microorganism (rad) is assume to be of cylindrical shape with hemispherical end
caps. Assuming that the cell density p [gIcm3] is constant, and assuming that the rodshaped organisms grow by increasing in length with Iittle change in the cylinder radius R
[cm] (axial, intercalary mode ofgrowth), the eell surface area for both morphologies are:

r
1

361r
S= ( - p'J.

m !3

S= 2m +~trR%
Rp 3

(cocci)

(53)

(rods)

(54)

However, in mast ofthe work done, the contribution ofthe end caps have been negleeted,
and therefore:

S=2m
Rp

(rods)

(55)

Eakman et al. [15] discuss how these relationships predict the cell mass - cell age
relationships for spheres and rods for a constant

{2}

and how these predictions capture

experimental evidenee obtained by others.

The cells are assumed to divide when they approach a certain critical mass or

48

division mass. [t is aIso assumed that this division mass follows a "Gaussian-type"
distnDution about a Mean division mass. However, since the cel1 mass cannot be less than
zero: the distribution is not Gaussian. The function for the specific probability of fission
for a critical division mass, 111c, derived by Eakman et al. [1 S] is given as:

(56)

where e [g] is a measure of the spread in the distribution of division mass around a Mean
division mass (me), and e/.J2 is the standard deviation of this distribution. Assuming a
single limiting substrate, Figure 13 shows the transient probability of cell division

r' (m, Cs)

as a funetion of cell mass m. The probability for ceU division increases with cell

mass, becoming very steep beyond the critical division mass. AIso shown in the graph is
the effect of the limiting substrate. When the substrate concentration decreases, the
transient probability ofcell division decreases for any given ceU mass, m.
The effect of the spread (s) about the division mass

lt1

can also be examined.

Figure 14 shows the transient probability of cell division r'(m,Cs ) as a function of cell
mass m for two different e values. As the spread about the division mass is reduced, the
increase in the transient probability of cell division becomes sharper because there is less
spread about the division mass 111c.

49

...--.
.e
,....., 25

--c
.--->
-~

U)

"-u

G)

20

C. 0.034 glL /

15
10

ca

..

.a
0

a.
c

...--0

U)

l!

1-

.
., ..
.. .
.
,
.
.. .
1

'0

----.a

..'"
0

0'

.' "

0.5

1.5

2.5

3.5

Cell mass x 10 12 (g)

Figure 13. Transient probability of cela division fi (m, Cs) venus cell mass m. The
graph illustrates the efTect of the limitinl lubstrate concentration. The values uled
in this plot were: me =3 X 10.12 1, E =4.242 X 10.13 1, J.1' =6 X 10.5 gI(cm1 hr), K. =
0.02 IlL, and De = 1 br [44].

so

.......,-c
--.->

80

60

.c:
0

Il

----a

...2
CD

90

70

.-.a---

e
a.

20

ca
.a

----..

10

f i)

c:

t-

=1x 10.

13

..
.,.

.
.

30

c
0

&

50

40

.
...
.

0.5

1.5

2.5

3.5

Cell mass x 10 12 (g)

Figure 14. Transient probability orcell division r'(m,C6 ) venus cell mass m. The
graph illustrates the etrect of the varyinl tbe spread 8 about the division masse The

values used in tbis plot were: me 3 S 10-12 1, Cs =0.034 gIL, J,l'

Ka = 0.02 gIL, .ad De = 1 brl [44].

SI

=,

10-5 gI(cm2hr),

During binary ceU division, assuming no loss of cell mass during the division
process, the mass of the parent cell must be divided between the two daughter cells.
Eakman et al. [15] proposed an expression for the density of daughter cell mass
distribution p(m,m'). They assumed that randomness exists in the partition of mass
between the daughter cells and that this randomness follows a Gaussian-type distribution.
They proposed:

m-!.1II.)1

_ _2_

p(m,m') =

(m')
s'lier[ -

(57)

2&'

where m' is the mass of the parent cell and e'1.Ji is the standard deviation of this
distribution. This expression is plotted as a function of daughter ceU mass in Figure 15.
Again the distribution cannat be Gaussian since the daughter ceUs cannot have a mass less
than zero or greater than that ofthe parent cell. The distribution of daughter cell mass bas
to be symmetrical about !.m' since
2

p(m,m') = pern'-m,m').

(58)

The efFect ofe' can alse be seen, where the smaller the spread in the distribution of mass
the narrower the distnoution.

52

...~6~-----------------------,
:: =
."o.
...
..
.
ft

"-"

::

1 x 10 13

.
! ..:
..
.
: .
:

..
C

.-:os
..a."
E

El

i :
:

..
~

f i)

f i)
fi)

el = 5 x 10 13

.0
Cen mass X 1012 (g)
Filure 15. Distribution of daulhter ceU DlalS for two difTerent e' values.

m' = 4 x 10.12 g.

53

Subramanian et al. [44] have solved the cell mass population balance equation for
various systems and conditions. Later, a different solution scheme will be derived ta solve
the model. Ta verify the solution, the simulation results will be compared with those
obtained by Subramanian et al. [44]. However in their simulations, Subramanian et al.
used a simpler relation for the distribution ofmass between the daughter cells:

') _ 30 m (m'-m)2
p (m,m .5

(59).

m'

This expression has no adjustable parameters and its graph is shown in Figure 16.
To simulate the SCF process, a substrate balance on the system must be
considered. For any arbitrary ith substrate (or produet) which enters and/or leaves the
reaetor through the feed and effluent streams, the following mass balance can be written:

(60)

where COli is the concentration of the idl substrate in the feed stream, Y t (m) is the fraction
1C

of component i in the mass taken up by the ceU [g of the ilh substrate 1 g of cell mass] and

r ; (m) is the fraction of companent i in the mass released by the cell (g ofthe ith substrate
~

1 g of cell mass]. Bath Y, (m) and

1C

ri- (m) depend on the physiologieal state ofthe eell

~

54

..

......
m
.......

0.50

..

0.45

0.35

)(

.-...0
~

.-...
.a
~

.fi)

~
fi)
fi)

0.40
0.30
0.25
0.20

ca

E 0.15

CD

0.10

r-

0.05
.1
~

=
ca

0.5

1.0

1.5 2.0 2.5 3.0

Cell mass X 10 12 (1/g)

3.5

4.0

Figure 16. Distribution of daulhter eeD mus a. liven by Subramanian et 111. [44].

55

and are therefore funetions ofthe cell mass. The SCF simulations also require that a mass
balance for oxygen be performed on the reador. This equation is the control equation
since the DO concentration is the parameter monitored for CYCDg. For any system,
assuming that the oxygen is sparged into the reaetor and that no oxygen is released by the
eell, the oxygen balance cm be written as:

(61)

where Co is the DO concentration in the reactor [gIL], Coin is the DO concentration ofthe
inIet stream [gIL], Co is the saturated DO concentration [gIL], kLa is the volumetrie
oxygen transfer coefficient [br-il and Y0 is the fraction of oxygen in the mass taken up by
x

the cell [g oxygen / g cell mass].

Equations (39), (60) and (61), along with the boundary condition (49) and the
initial conditions W(O,m), Ca(O) and Co(O) constitute the fully defined cell mass model for
the continuous (0 < e < CX and batch (9 =oc) reaetor problems. Later it will be seen how
these equations are modified to simulate the SCF process.
Eakman et al. [1 S] also presented a discussion on the relation of the cell mass
model to the segregated unstNetured model (total il density) and the distributed model
(viable biomass concentration), in addition to the relation between the cell mass model and
the eell age model. It is aIso worth noting that the viable biomass concentration C [gIL]
CID

be obtain ftom the cell mass model by taking the first moment of the cell mass
56

distribution:

GO

=l mW(t,m)dm.

(62)

3.3 Solution Scheme

The system of equations which have to be solved consist of one non-linear, partialintegro differential equation (population balance equation), coupled to two non-linear
differential equations (limiting substrate and oxygen balances). The cell mass population
balance model has been solved by Subramanian and Ramkrishna using the method of
moment equations along with the Laguerre function expansion [35,43]. Other techniques
used to solved the general population equation for particles undergoing a cambination of
growth, comminution, and collection are reviewed by Ramkrishna [37]. More recently,
Liou et al. [29] has obtained the solution to the ceU mass population balance equation
using a successive generations approaeh.
This work uses the Galerkin Finite Element Method [17,28] along with the implicit
predictor-corrector Euler scheme [17,19] to solve the microbial population model.

3.3.1 Galerki" Fillite Element Met1lod

To solve the eeU mass population balance equation (39) for the ceU mass
distribution, W(t,m), the foUowing trial solution is defined:

...

W(t,m)

=~wJ(t)8j(m)

(63)

j-I

57

where

W(t,m)

is the trial solution for the cell mass distribution, Wj(t) are unknown

funetions oftime, Sj(m) are known nearly orthogonal basis functons, and N is the number
of Dodes in the mesh spanning the cell mass domain 0 S m S 1JJmq, where m....x is the upper
cell mass limit above which, for ail practical purposes, no cells exist. By substituting this
trial solution into equation (39), the residual R was defined as:

R =W(t,m) + blr(m,C.)W(t,m)] 2fCIJ r (M ,C.)W(t,m' )p(m,1d)dnt

li

an

(64)

".

+G +[" (m,C.> + 0(m, C. W(t,m> * 0

The residual is a measure of the error occurred when the trial solution is substituted into
the cell mass population balance equation. The problem lies in obtaining the funetions

Wj

that minimize the residual. This is done by setting the inner product of the residual and of
a set ofweighing funetions equal to zero:

(65)

where ~i are the weighing funetions, and Dlaaax is the upper cell mass mit over which the
finite mesh is defined. To find a numerical solution to equation (39), the mesh had to be
defined such as to cover the entire domain over which the cell mass distnDution has a nonzero solution. Applying the Galerkin method, the weighing funetions were set equal to the

58

basis funetion such that:

(66)

In addition, the population balance equation is also coupled to the miting

substrate balance. In the foUowing simulations, a single limiting substrate will be assumed.
This assumption was also foUowed by Wincure et al [53]. Similarly, the oxygen balance
equation is coupled to both the population balance equation and the limiting substrate
equation. Therefore, a total of N + 2 unknowns must be solved in N + 2 equations.
Rewriting this system of equations in veetor notation yields:

F~)=O

(67)

f. Cl)
f'l~)

w1
w2

f3<l.)

w3

where F<l.) = .

!N<l)

S<l>
DO(y)

,l= .

(68)

wN
Cs
Co

59

JI

ras- R. 9 dm =m.. W(t,m) /J dm+ m- bll(m,Cs )W(t,m)] /J.dm

Jo

-T(

an'

21r'(m' C.)W'(t.ml)p(m.m')'in)e,cbn

TG

fo

i = 1.2...N. (69)

+r'(m,c.)+9(m,c.)W(t.m)8,cbn=o

dC --cc;
1
S=-'
-C,)-ICIO [ ys(m)'"(m}-Ys(m)" (m,C,)] W(t,m)dm=O,
dl IJ
-x
o -x

(7D)

and

(71)

The solution vector

of equation (67) was solved using the Newton-Raphson

iteration scheme [17,19]. For the vector equation, this scheme may be written as:

(72)

where k is the iteration index, and -L is the Jacobian matrix:

(73)

60

This iterative scheme can be rewritten as:

yk+l
_

= yt
_ _ J If -1 F(yt).
_

With each

iteratio~

the veetor

(74)

~t+l

converges quadratically towards its true value. The

iteration is carried out until the difference between successive solutions reaches a value
below a user specified tolerance.
Chapeau basis funetions (Le. the function 9j in equation (63 were used ta solve
the cell mass population balance equation.

These funetions are linear and nearly

orthogonal in that they "hardly" overlap. Therefore when evaluating the integrals in~, the
integration limits may be reduced to values covering the range where ai is non zero. In
addition, over each element, there are only two contributions trom the basis funetions.
This reduces the likelihood of having to solve ill-conditioned matrices. The integrals were
solved using a 3-point Gaussian quadrature method [19].

3.3.2 Predictor-Corrector E"ler Scheme

The solution to the N + 2 equations discussed above must be found as a funetion
of time. This was accomplished using the implicit Predietor-Correetor Euler scheme
[17,19]. This numerical method consists of two step. The first is an explicit predictor
step in which a solution is approximated tram previous known solutions.
Using the notation developed in the previous section, the predictor $lep can be

61

written as:

, =Y + At ( y~ -y
:..11-1
Y~+1:..11
11+1
M

(75)

"
where l

Pn+ 1 is

the predieted value ofthe dependent variable veetor l,

known values of the dependent variable at time ln and

the user specified time step from tn ta

tn+l

t... l

ln

respeetively,

and l

Atll+1

n-I

and

are the
Atn

are

and to-I ta ln respectively, and n is the solution

index. The term in the bracket is the first arder difference approximation of the tirst
derivative of l at time tn
The second step consists of an implicit procedure which corrects the predicted
value lPn+ 1 to yield a more accurate solution for ln+l. This procedure uses the predieted
solution lPn+ 1 to estimate the tinte derivatives at tn+l:

_i

li

=ylf+l _ y"
1

(76)

Atlf+l

This approximation, along with the predieted value y Pa+1 is then re-substituted back into
equation (67) and the Newton-Raphson iteration scheme is used to find the correeted
solution ll:n+l at time tn+l. If the absolute ditrerence between the predieted and correeted
values is greater than a user-speclfed tolerance, in this case Il cn+l -

lelll ~ 1 x 10", the

solution is rejected and the process is repeated with a smaller time step. If this di1ference

62

is within the specified tolerance, the solution is accepted and the process is continued to
find the next solution Ytt+2. Ta veritY for mesh independence, the number ofnodes where
increased unt no change in the solution could be noticed. The number of nodes used for
the SCF simulations was 61. The distribution of nodes were accommodated as to be
concentrated in the space spanned by the cell mass distribution W(m,t).
3.4 Results and Discussion

3.4.1 Verification ollie Solution

In arder to verny the validity of the solution scheme, the cell mass population

balance was solved, using the new solution scheme, for the chemostat (9 > CXl) and batch
(8 = (J) problems. The model for the chemostat and batch fermentation is defined by
equations (39), (60) and (61), with boundary conditions (49). The simulation results were
then compared to the results obtained by Subramanian et al. [44]. Table 1 shows the
parameter vaiues, taken tram reference [44], that were used for these simulations. The
organisms were assumed to be bacilli with cylindrica1 radius R. No cell death was
assumed. The distribution ofdaughter cell mass WBS given by equation (59).
In arder to study the behavior of the cell population balance model, Subramanian

et al. [44] used three different sets of initial conditions to simulate the chemostat reactar.

These initial conditions are given by the foUowing equations:

63

Parameters

Values

IDe

3 x 10 g

5 X 10.5 cm

K.

0.02 WL

J.1

1 br-1

J.1

6 X 10.5 gI(cm2.hr)

y..

0.75

JI

Y ..
z

3''-12 x 10.13 g

1.01 g/cm3

C.o

2.5 gIL

kLa

300 br-1

6.7

Co

0.2624 g/L

Table 1. Parameter values used for the simulations of the chemostat and batch
modell.

64

..

m --

W(O,m) = -e &10 24

(77)

w(o,m)=o.ot(:re :10'"

(78)

W(O,m) =

o.{:)e :10"

(79)

Equation (77) represents an initial cell mass distribution composed of organisms of

relatively small cell mass, while that of equation (78) represents a broader, more uniformly
distributed cell mass distribution. The initial condition given by equation (79) has the
same distribution as equation (77) except that the total cell number has been reduced by a
factor of 10. These tbree sets of initial conditions lead to three very different solutions to
the chemostat case, as is observed from the simulation results in Figures 17 to 22. These
graphs plot the DO concentration, the limiting substrate concentration, the biomass
concentration, and the cell number concentration as a function of time. The evolution of
the cell mass distribution is also shawn. The cell number concentration profiles presented
here are normalized with respect to the parameter

E.

This was done to facilitate the

comparison of the results obtained in this thesis with those published by Subramanian et
al [44]. The solutions obtained by the new solution scheme developed in the present

thesis reproduce those published in the Iiterature [44]. A discussion ofthese results is also
given in tbis reference.

65

0.7 ~-------------r-0.265

0.260

0.6

0.255
Lim iting Su bs trate

...... O.S

0.250 ......
~
~
0.245 =

~ 0.4

.=

0.240 ~

Dissolved Oxygen

(Il

.c

= 0.3
.-== 0.2

0.235

ri.)

DQ

0.230

.-E 0.1

0.225 Q

0.220

0.0
1.8

~-----------_....-&0.215

1.8
Cali Number

1.6

...

~
1

o
1.4
~
~w ...... 1.2

1.6

1.4
Biomass

=
0

1.2 ;
c

x~

.. i 1.0

1.0

u=
~u
E u 0.8
:::s-~ 0.6

u 0.4

0.4

0.2

0.2

0.0

0.0

fic~
......
o~

u .!!
en
0.6 :1
0.8

0.0

1.0

2.0

3.0

4.0

iD

5.0

Tlme (hours)
Figure 17. DO, limitiDllubstnte, biomul, Ind ceU Damber prordes for a
chemoltat, UliDI the initial conditions liven by equatioD (77).

66

0.40 . . . . . . . - - - - - - - - - - - - - - - - - - - - ,
1.8

t=O hr

::::i 0.35
......
en

~ 0.30
~

1
1

0.25

0.20 1-

0.15

CIl

;. 0.10

!
LI.

0.05 ,
0.00

-II,~-__+_--__4_----::~~
.....__+__--_+_--~

234
Cell Mass (10 12 g)

Figure 18. Cell mass distributions for a chemostat, usinl the initial conditions given
by equation (77). The dasbed line represents the study state distribution.

67

0.6 - - - - - - - - - - - - - - - - - - - - - - 0.270

o.s
~
:y
0.4

-- 0.260

-~

-- 0.250

-~

-0.240

-~

0.230 0

.!

Dissolved Oxygen

jO.3 --\ r-------~

1:

----...eri 0.2 --

-- 0.220 -;

~ 0.1 -~

Limiting Su bstrate

-~

0.0 .........-------------~
2.5

0.210

0.200
2.5
2.3

2.0

2.1

Cell Number

Q
.....

-)( :i1.5

C
0

.-....

1.9 ....
I!
c

1.7

.. .....
en
CD.ae cu

1.5

CI
U....-

C..J
0 Cft

u ......

::s ~1.0

1.3

1)

1.1 E
o

-z

II)

1ft
ftS

0.5

0.9
0.7

0.0

----.....-.---~--'-----I--~ 0.5

o
Figure 19.

234
lime (hours)

iii

no, limiting substrate, bioDlIIS, aad ceu aumber profiles for a

cbeDlostat, usinl the initial conditioDs liven by equatioD (78).

68

0.50 - r - - - - - - - - - - - - - - - - - - - - - .
~O.45
..J

-~

iJ 0.40
0.35

.;-'
~ 0.30

;; 0.25
~0.20

cr

0.15

! 0.10
iL

0.05

0.00

~~-+-----t__-__t-~__+_--__+_.::::==~

234
Cell Mass (10.12 g)

Figure 20. CeU mass distributions for a cbemostlt, usinl the initia. conditions given
by equation (78). The dashed line represents the steady state distributioD.

69

1.6

0.27

1.4

Limiting Substrate

0.26

. 1.2

0.25 ~

:i

~ 1.0

.i~

0.2

~
0.21

0.0

0.20

2.5

2.5

2.0

2.0

0.22 "0

-~~1.5
1

c
0

..

;;

l!

Cell Number

1.5 CD
u .....

.=
II.

0.23

0.8

0.6
u
c:
0.4
~

ii

0.24

Dissolved Oxygen

en

C-I

ua
....

J:a CD

E ~1.0
::1

1.0 ..
(1)

CD

0.5

E
0.5 ii5

0.0

0.0

u
Z

6
lime (hours)
4

10

Figure 21. DO, limiting substrate, biomass, Ind ceU Bumber pronles for a
chemostat, using the initial conditions gjven by equatioD (79).

70

0.50 . . , - - - - - - - - - - - - - - - - - - - - - - - ,

.......... 0.45

4.8

-"8fi 0.40
0.35
~

~ 0.30

c
... 0.25
)(

~0.20

0.15

...~0.10

~ 0.05

......_4_--t__-~

0.00 ~c=+~~~=~~

234
Cell Mass (10.12 g)

Figure 22. Cell mass distributions for a chemostat, usinl the initial conditions given
by equation (79). Tbe dasbed line represents the steady state distribution.

71

The case of the batch fennentation revealed a mathematical inconsistency between

the population balance, as described by Eakman et al [15], and the simulation results
obtained by Subramanian et al. [44]. The simulation results, in reference [44], for the
batch problem show the fermentation proceeding up to the exhaustion of the limiting
substrate. At low limiting substrate concentrations, the single cell growth rate is allowed
to become negative, indicating that the ceUs are experiencing a net mass loss. In faet, the
single cell growth rate attains a negative value when

(80)

This is also true for the transition probability funetion for cell division ['(o1,C.), which
leads ta the inconsistencies between the published simulations and the results obtained
here. These results for the cell number profile and the biomass concentration are seen in
Figure 23.
This profile is very difTerent from that obtained by Subramanian el al. [44] for the
same conditions. Figure 23 shows the cell number profile decreasing in time once the
miting substrate reaches a concentration below that specified by equation (80). Since no
cell death was assumed, this decrease should not be predieted by the model. The negative
['(01,C.) function eventually causes eeUs to be lost ftom the eeU mass mesh and ceeates a

sharp profile in the ceU mass distnDution. This causes the steep deerease in the cell
density, which is allowed ta attained negative values.

72

2.5 , . - - - - - - - - - - - - - - - - - - - - - . ,

......

-u~ai

2.0

....... 1.5

1.0~---

_..... 0.5
1

CA)

>< 0.0
~

-0.5

-1.0

i
u

-1.5

-2.0 --------------------+-------'
2.5 - - - - -

......
....
.......

Cft

2.0

~c

1.5

1.0

cu
u
c

fi)
fi)

al

E 0.5
o
iD 0.0

0.0

__..1

010_

0.5

1.0

---"

1.5

.......

2.0

Tlme (hours)
Figure 23. Solution of the ceU mass populatioD model, u given by the equatioDs in
reference [15]. Tbis figures shows the brealulown of the modellate in the
fermentation.

73

The biomass concentration decreases beyond the concentration specified by

equation (80). This decrease was also predicted by Subramanian et al [44], and reflects a
net release of mass by the microorganisms.

However once the cell number attains

negative values, the biomass concentration is seen to increase very rapidly, reflecting a
breakdo\vn in the model.
Mathematically, allowing r'(rn,c.) to reach negative values is equivalent to
allowing two cells of smaller cell mass to come together to form a single, larger organism.
This process is \'ery similar to the aggregation phenomenon seen in particulate systems.
However, there is no biological mechanisms for this type of aggregation in microbial
systems. The simulations obtained by Subramanian et al. [44] do not predict a decrease in
cell number. Vet, they do not discuss this irregularity.
Although this discussion highlights a major breakdown in the model, this
irregularity oceurs ooly beyond the stationary growth phase. In SCF, the system is made
to cycle upon the detection of the minimum in the DO concentration. This minimum
occurs before the onset of the stationary phase. Therefore the breakdown in the model
occurs outside the bounds of growth phases observed in SCF, and the population balance
equation can still he used ta model the system, without any modification. If this was not
the case modifications would be necessary. A modification that would overcome this
breakdown is obtained by setting the transient probability of division function to zero for
substrate concentration below that specified by equation (80).

The results of

implementing tms modification for the simulation of the batch fermentation are shown in
Figures 24 and 25 for the initial conditions of equation (77). Figure 24 show the DO

74

concentration, the limiting substrate concentration, the biomass concentration, and the cell
number profile as a function of time. Figure 2S shows the evolution of the cell mass
distribution.

The simulations results obtained for the batch fermentation using this

modification matches thase obtained by Subramanian et al. [44] for the same conditions.

3.4.2 Application oftire Cell Mass Population Balance Model to the SCF Problem
The ceU mass population balance model given by equations (39), (60) and (61),
and boundary condition (49) was modified ta simulate the SCF process. Equations (81),
(82) and (83) show the modified equations.

W(t, m) + [r(m,C,)W(t,m)] = 2 r' (m' ,C,)W(t, m' )p(m, m' )dm'

t
am
m
CIO

_[1 (m, C,)W(t,m) -

L jW(/, m)c5(t - tmia01tj)

(81)

j=1

de
-j-= -IYi. (m)r(m,C.)W(t,m)dm + L[JO(t-t....o,)(C: -C.)]
CIO

CIO

~1

(83).

CIO

L [/6(t - t
)-1

(82)

rainO :

)(C~

- Co)]

7S

2.5

0.30
Dissolved Oxygen

0.2S

2.0

:i
~

--f
~

0.20 8

1.S

O.IS!

.!

= 1.0
.-.....-ec: 0.5
~

0. 10

Umiting Substrats

0.0
2.5 .

0.00

..

.-1
w

)( 31.5
......
~

Il

2.0
1.8
C
1.6.2
1.4 ~
C
1.2 ~ ~
C..I
1.0 0
CJ ...
0.8 .,

2.0

rot

Biomass

fil

.au
z

O.OS

:1

-l1

a.

~1.0

.,

0.5

0.6 ;
0.4

0.0

0.2
0.0

il

0.0

0.5

1.0

1.5

2.0

nme (hours)
Fipre 24. Results from the modified population balance equatioD for the DO,
Iimitiog lubstrate, biomass, and ceU Dumber profiles of a batch reactor, using the
initial conditioDs liven by equation (77).

76

0.7

i r

1.4

0.6

CD

C'1

0.5

c 0.4
~

><

0.3

CD

0.2

~
c

=
cr

! 0.1

&1.

0.0

~--+----+--=====~~---~---4---~

234

Cell Mass (10-12 g)

Figure 25. Results for the modified population balance equatioD for the ceU mass
distributions of a batch reactor, using the initial conditions given by equation (77).

77

As in the case ofthe batch fermentation,

eis set equal to O.

In addition, upon deteetion of

the minimum in DO, the cell mass distribution at that moment is multiplied by the fi1l /
empty fraction of ..!., simulating the removal ofhalfthe mixed broth trom the reaaor. The
2

effect of emptying and refilling the reaetor, on the population number, is accounted for by
the summation term in equation (81). Similarly, the equations describing the limiting
substrate and DO concentrations were also modified to account for the cycling of the
system (equations (82) and (83), respectively). Agam, the distribution of mass into the
daughter cells were described by equation (60), and the organisms were assumed to be
bacillus of radius R. Table 2 lists the parameter values estimated from the work of
Wincure [53]. The intracycle solutions were obtained usmg the solution scheme discussed
in section 3.3.
Figures 26, 27 and 28 show the results of applying the cell mass model to simulate
the SCF process. Figure 26 shows the biomass concentration (top), the Iimiting substrate
concentration (middle), and the DO concentration (bottom) profiles predieted by the
model. A comparison ofthese resuIts with the simulations obtained by Wmcure et al. [53]
show that the cell mass population balance Model was able to capture the macroscopic
profiles of the system. The model was able to capture the DO concentration Co, the
Iimiting substrate concentration CI' and the biomass concentration C as a function of time.
The stable periodicity, along with the cycle length was also captured by the model.
Figure 27 shows the curves for the cell number (top) and the DO (bottom)
concentrations as a funetion of time, obtained from the simulations. The top part of this
figure suggests that the cen population predicted by the model is not

78

synchronized~

The

Parameters

Values

l11

3 x 10 g

5 x 10-5 cm

K.

1 x 10-' gIL

J.1c

o br-l

Il

1.515 X 10.5 gI(cm2br)

Y,

1.31

Y,

&

3~2

10.13 g

LOI glcm3

CIO

0.6246 gIL

kLa

145.2 br-1

Y"-

5.45

Co

0.0371 gIL

Table 2. Parameter values estiDlated (rom the work orWincure et aL [53).

79

500

-r---------------....,

450
400
~350

-1

r300

250
2200
.2
ID 150
100
50

0""""'----------------350

-r---------------,

_300
"al

..J

E250

~200
fi)

J:l

a150
al

i100

:J 50
0""""----..........- - - - - - - - - - - - - - - - - - . .

32

-r----------------,

20 + - - - _ r - - - - - + - - - - + - - - - - t
7
9
15
11
13

lime (hour.)

Figure 26. Riomasl, limiting lobstrate, aad DO concentntion prordes (or the SCF
systeDl.

80

.J

2.5

li
u

:'2
~

Ct

;1.5
~

!= 1

~O.: J
32

--

-'

30

28

26

CIl
~

-a 24

15

..-

:>

( ft

22

20
7

11

13

15

lime (hours)

Figure 27. Cell number (top) and DO (bottom) profiles for the SCF system.

81

90

......

80

-' 70

---"

-......u

60

50

..

...
0

M
~

u
c

a~

..
.,
...
..
..
..
.,

40
30

20

l! 10

0
0

246
CeU Mass (10 12 g)

Figure 28. Cell mass distribution for the SCF system (solid). The dasbed line
represent a tell mass distribution for an asyDchronized continuous culture.

82

cell population is seen to double exaetly once during each cycle, however, the cell number
increases exponentially throughout the entire cycle. This exponential increase refleets an
asynchro~ed

cell population. The experimental profiles show a step increase in cell

number for each cycle.

Figure 28 shows the cell mass distribution which results ftam this simulation. The
dashed line represents an asynchronized cell mass distribution. The dashed distribution is
the steady state cell mass distribution from the chemostat simulations shawn earlier,
amplified to he on the same scale as the distribution obtained ftom the SCF simulation.
These results show that the microbial population equation is able to capture the
macroscopie features of the system, but is unable to predict the synchronized cell
population, as shawn in the reported experimental results (see Figures 2 to 7).
A eloser look at the chemostat simulations shawn earlier (Figures 17 to 22), that

were repeated based on the work of Subramanian et al. [44], revealed that the steady state
cell distribution was independent ofthe initial cell mass distribution imposed on the model.
This retleets the dispersive behavior of the system. This dispersion causes any pattern in
the initial cell mass distribution, other than the steady state distribution, to be lost as the
cells replicate. Therefore, in arder for the microorganisms to converge into a common
point within the cell mass distribution and obtain a synchronized culture, a mechanism
must be incorporated into the model to overcome the dispersion etfeet inherent in cellular
division.
It is worth noting that the term dispersion, in this context, reCers to the randomness
which is exist in the division process. That is to say, microorganism are assumed to divide

83

when they approach a critical division mass. Therefore, there exist a distribution about
tms division mass where cellular division take place. The faet that not all cell divide
exactly at the division mass leads to a dispersion in the cell mass distribution. The effeet
ofdispersion will be illustrated further in the next section.

3.4.3 Dispersion Elfects witlain the Cell Maas Population Balance Model
The chemostat simulations shown in section 4.3.1 show that the irtial cell mass
distribution profile is modified within one generation time. This is due to the fact that
cellular division of microorganisms does not occur exactly at the division mass, but rather
takes place about a critical cell mass, following a "Gaussian-typett distribution. Similarly
the model also allows for unequal distribution of mass into the daughter cells. The spread
ofthe distribution ofcell division about the critical division mass is govemed by the term E
in equation (84), and the spread ofthe distribution ofcell mass iota daughter cells is set by
the term E' in equation (85). Both these expressions were described in section 3.2.

(84)

p(m,m ) =

(ml)
EI.J;erj -

(85)

2&1

These equations account for the randomness which is assumed ta exist in the division

84

process ofmicrobes.
In order to examine the effect of lhis dispersive effect, the randomness which is

assumed to exist in the division and birth processes was reduced for the Dext simulation.
The parameter values used in this simulation were those given in Table 1 except that the
measure of spread of division mass,

B,

about the critical cell mass, 111c:, was reduced by a

factor of 4. Similarly, the randomness in the partition of mass between the daughter cell
was reduced. Equation (8S) was used to described the distribution of mass into daughter

cells. The measurement of spread in daughter cell mass distribution, &', was set to O.S x
10.13

To study the effect of dispersion, a very narrow initial cell mass distribution was
used for tbis simulation. The top graph of Figure 29 shows the cell number and biomass
concentration profiles.

Again, the cell number was normalized with respect to the

parameter &. The bottom graph shows the initial (solid line) and the steady stale (dashed
line) cell mass distribution.
The effect of the narrow initial distribution can he seen in the cell number profile.
Organisms cao only. divide when they have reached the Crticai mass for division. The
narrow initial distribution will therefore have to travel to the right along the cell mass
domain, in order to reach the division mass. During this period, no cellular division should
occur, and the total cell count should remain constant. Upon reaching the division mass,
the entire cell population is seen to double in a very narrow time interval, causing the steplike increase in cell number. This process is repeated and results in the step increase in cell
density. The abruptness of the step increase is gradually lost as the randomness which

85

1.0

---------------~

0.4

c
o

0.3

0.2

t
Bc.J
0

....
g

"iSt

(Jfit

0.1

0.2

ii5

0.0 + - - - + - - - + - - - - t - - - - + - - - - + 0.0
0.0
0.2
0.4
0.6
0.8
1.0
lime (hours)
0.16

~--------------~

i
.-,

8 0.12~

\'\.

ca
;; 0.08

~
c:

104

",

.\.

"

.....

......\\

) '\

1&.

0.00 + - - - - + -.........-"""'-~---t--------i

C811 M (10 -12 g)

Figure 29. Cell Bumber, biomass, and celllDlsl distribution profiles for a chemoltat
reactor, BIing a narrow initial ceU mail distribution. Tbe fullline il tbe initial
narrow cell mass diltribution It time t = o. The dashed line is the ceU ma
distribution tbat devetops u t --. CD.

86

exists in the division and birth processes aet ta reduce the sharpness of the initial cell mass
distribution. Within a few generation times the initial distribution (solid line) broadens.
The steady state distribution is shown on the graph as the broader cell mass distnDution
(dashed line).
Reducing the randomness in the division process resulted in a very different steady
state cell mass distribution when compared to the distribution obtained trom the previous
simulations. The resulting cell population spanned a smaller region in the cell mass space,
from a cell mass ofapproximately 1.S x 10.12 g to about 3.0 x 10.12 g. The previous steady
state cell mass distnoution had a span tram 0 to approximately 4.0 x 10.12 g. The end of
the distribution is also shaper than those ofthe other distributions discussed earlier. These
sharper profiles required the refinement of the cell mass mesh, at the distribution
extremities, in arder ta eliminate oscillations in the solution.
Reducing the spread in the division and birth probability funetions also allowed for
a skewness in the cell mass distribution ta be observed. This skewness arose due to the
faet that the single cell growth rate foUowed a tirst order process. Therefore, as an
organism increased in cell mass, its mass growth rate also increased. The larger organisms
traveled faster along the cell mass axis. This acceleration in growth rate caused the ceU

mass distribution to be skewed towards larger cell masses. This skewed profile was
masked when the random effeets were large.
3.5 Modification to the Cell Ma Population Balance Model

The eeU mass population balance equation (81), proposed by Eakman et al. [15],
was nat able ta capture the feature of il synchrony when used to model the SCF

87

proeess. In the eell age model, a feedhaek mechanism between the critica1 division age
and the growth environment allowed for the synchronization ofthe microorganism when a
forcing function was applied to the system. In the case of the present cell mode~ no such
mechanism is possible. The control of cell division is assumed to depend on the critical
division mass Mc, which was held constant (sec equation (84) or equation (56) in section
3.2).

Experimental evidence, however, suggest that a strong correlation can exist

between the control of cellular division and the growth rate [2,7,10,14,16,27,32,47,51].
More specifically, the average eell mass or cell size of a growing population of
mieroorganism is often seen to be proportional to the growth rate.

For example,

Nanninga and Woldringh [32] discuss the relationship between the average cell mass, and
the growth rate of the culture. They aIso give experimental evidence ofthe dependence of
cell mass, volume, surface area, length and width, on the 810wth rate of the
microorganism.
Similarly, Donachie [14] had previously reported the relationship between eeU size
and the time of initiation of DNA replication. He had determined that the mass at which
initiation took place was always an integral multiple of a partieular mass. This property
was also used to expIain the increase in ceU size with increase in growth rate.
It is clear that the control of cellular division involves Many complex regulatory
pathways, however eell mass often remains a good indication ofthe progression ofthe cell
through the cell cycle [2,7,10,27].
Such feedback mechanism between the growth environment and the critical eell

88

mass for division have been used to model various systems. Hjomo and Bailey [24] use
such a relationship between the growth rate and the cell mass at which a cell became
committed to division, to capture the transient response of a budding yeast population to a
shift in growth rate. Similarly, such a relation bas also been used ta model spontaneous
oscillations in continuous cultures ofyeast [6,25,30,34].
Other experiments shed more light on the relationship between division mass and
growth rate. Cutler and Evans [9] condueted experiments in which they sampled a batch
culture al ditrerent points of the growth curve. Each of these samples were then used ta
inoculate new flasks. The flasks which were inoculated with cells taken at the onset of the
stationary phase were found ta be synchronous. These experiments were condueted with
Proteus vulgaris and three ditrerent stains of Escherichia coli, and ail resulted in
synchronous cultures. The cell synchrony wu eventually lost after a few generation times
due to the random effeets associated with the division process.
Another set of experiments by Wachi and Matsuhashi [51] a1so demonstrate the
particularity of the onset of the stationary phase on the control of cell division. Their
work consisted in the study of negative control of cell division by a gene responsible for
determining the rad shape of E. coli. In this woele, experimental evidence was gathered
which shows an acceleration in cellular division in the late exponential ta early stationary
phase. During tms period, it was reported that the cell population consisted of smaller
rods. This later evidence could be explained by a reductionin the critical division mass
just prior ta the onset ofthe stationary phase ofgrowth. This reduetion would result in an
acceleration in cellular division and in a reduetion of the average cell size of the

89

population. Simi1arly, this division mass profile would also result in the behavior observed
by Cutler and Evans [9]. Upon the onset of the stationary phase, the cell population
would be composed of smaller microorganisms. Reinoculation into fresh medium would
result in an increase in the growth rate and therefore in division mass. In order to divide,
the microorganisms would have had to grow to the new division mass. The resultant cell
density profile would consist of step increases characteristic of a narrower cell mass
distribution.
Figure 30 shows a possible profile for the relationship between the critical division
mass and the limiting substrate. For large substrate concentrations (larger growth rates)
the critical division mass remains constant. Just prior to the onset of the stationary phase,
the division mass is aUowed to decrease to a minimum value. This division mass profile
will be used in the foUowing simulations.
3.& Resulta and Discussion of the Modifled Cell Mass Madel
ln arder ta account for the effect of varying substrate concentration on the cell

division cycle, the critical mass for division

Ille

wu allowed to vary with changes in

growth environment. Figure 30 depicts the behavior of the critica1 division mass as a
funetion of limiting substrate concentration. The profile assumed that the average cell size
remained constant for higher limiting substrate concentrations, corresponding to the
maximum growth rate. Belowa concentration of 0.15 g/L, the critical division mass was
assumed to decrease linearly fram its maximum value of3.8 x 1012g to its lower value of
3.0 x 10.12 g. Belowa substrate concentration of 0.02 g/L, the division mass was assumed
ta remain constant at its lower limit. The remaining parameter values were the same as

90

--....
cd

.....,
.....
...

Limiting Substrate, Cs
Filure 30. Critieal division mass me as a Cunction oC limiting substrate C.. The
panmeter me appean in equation (84) and is shown to atrect the degree of
synehrony predicted by tbe model (see test).

91

those used in section 3.4.2, and are listed in Table 2. Although a SRlooth profile couId
have been used, the profile used here captures the required funetionality of lI1c: on C.
The effect of imposing this scheme on the total cell number profile is seen in the
results shawn in Figure 31. In this graph, the cell density concentration is plotted as a
funetion oftime. Within each cycle, the cell number is seen to remain constant for the tirst
part of the cycle, followed by a sharp increase towards the end of the cycle. Figure 32
shows a comparison between the cell number trend of this simulation (top) and those
obtained when the critical division mass lt1c: is held constant at 3.0 x 10.12 g (bottom). The
effect of introducing the relationsip between division mass ll1c and the limiting substrate
C. is seen in tms figure. By aUowing the critical division mass D1e to decrease with growth
rate r(m,C.), the total cell number profile approached those observed in the experiments
(Figures (2) to (7.
This simulation of SCF predicted total ceU number values al the end of each cycle
that were lower than those obtained when the division mass was held constant. This is
due ta the faet that in the present simulations, the critical division mass lt1c was given an
upper value of 3.8 x 10.12 g when the microorganisms were growing at the maximum
growth rate. The eeUs were therefore allowed to reaeh a larger ceU mass when eompared
to the previous simulations.

Since the biomass concentration as a funetion of time

remained the same for both simulations, the ceU number profile predieted by the new
model was slightly lower in value.
Figure 33 shows the cell mass distribution prior and after the shift in the critical
division mass. The decrease in the critical division mass results in a shift in the cell mass

92

3.0
.....
..J

i-

2.5

u
.......
, 2.0

~
~

'P

1.5

CI
.a
e 1.0

:::1

-cu 0.5
Z

0.0
14

15

16

17

Tlme (hours)

Figure 31. Simulation mults of ceU number 1. a function of time for tbe SCF
system, witb ceU mass division controL

93

-=i

3.0

_ 2.5

CJ
.....

2.0

)(

1.5

E 1.0
~

- 0.5
Qi

(J

0.0
3

o
;

1.5

~
cfil

==

0.5

O...J..------...&....-----.. . . . .- -------J
14

15

16

17

lime (hours)

Filure 32. Simulation reluits for the ceU Bumber profile of the SCF system. Top:
profile obtained when the division mais decreases witb limitinllubstrate. Bottom:
protile obtaiaed wben tbe division

94

mUI DIe

is beld CODstant.

distribution towards smaller cell masses. Since the width of the distnDution, assuming no
random etrects, is equal to 1/2 the division mass, a decrease in the division mass causes a
narroWng in the cell mass distribution (solid distribution in Figure 33). Eventually the
minimum in DO is detected and the system is made to cycle. The addition of fresh
medium into the reaetor will cause an increase in the division mass.

The narrow

distribution will therefore have to travel farther towards larger cclI masses in order for cell
division to oceur. This period ofno cell division results in the constant cell number profile
which is observed early in the cycle.

Upon reaching the new division mass, the

microorganisms will double in a narrow time interval, causing a sharp increase in cell
number lowards the end ofthe cycle. It is worth noting that this mechanism for synchrony
is quite differenl from the induction mechanism proposed by Hjonso [23]. Although both
these mechanisms involve a shift in the location ofcellular division, the cell age mechanism
is such that ail cells start their new cellular cycle at the same point after division. That is
ta say ail newbom cells acquire a cell age of zero. Therefore, under certain conditions,

lhis mechanism can lead to infinitely narrow cell age distnoutions. In the case of the ceU
mass model, the newbom ceUs acquire a cell mass of 1/2 the cell mass at division. The
order or arrangement of the cell within the distribution will not change.

Two

microorganisms with ditferent cell masses will Dever attain a similar cell mass at any given

time, barring the effeet ofrandomness.

The aetual division mass profile MOst likely would not follow a linear decrease as
shawn in Figure 3O. This trend wu selected to ret1ect the decrease in division mass with
limiting substrate concentration. The aetual profile would probably exhibit a smooth

9S

1.6 - r - - - - - - - - - - - - - - - - - - - - ,
~

:::!
en

-Qui

1.2

~ 0.8

~
c
CIl

a-

'. "

0.4

l!

...
.....................

"'..

LI.

o. 0

-+-----o+---",,;JtI--"-I-------+~-_-.:..+---~

Cell Mass (10 12 g)

Figure 33. Effett of tbe varyiDI critical division masi Die on the ceU mUI

distribution. The dasbed line is tbe cell mass distribution prior to the sbilt of the
critical division Dlass. The soUd line is tbe eeU mUI distribution after a sbift in tbe

eridtal division mUI.

96

transition between the constant critical division mass regions and the transient domain. A

smooth transition would lead to a smooth rise in the ceU density profile.
Similarly, the upper and lower values of the division mass were selected as to
simplify the solution scheme. Allowing the critica1 division mass to vary over a wider
range would result in a longer period of constant cell number concentration within each
the cycle, and a sharper increase towards the end of the cycle. However, domg sa
requires an adaptable mesh scheme in arder to avoid redundant nodes, which lead to
singularities in the Jacobian matrix upon the inversion procedure.
Cutler and Evans [9] had observed that cells growing in the substrate rich medium
had an average cell size up to four times greater than the cells in the stationary phase. In
this simulations, the cells growing in a high substrate concentration were 27 % larger than
the cells growing in a low substrate concentration.

Nevertheless, the general trend

assumed here, along with the location of the deerease in the critical division mass does
refleet the experimental observations.

3.7 Conclusions
The cell mass population balance equation proposed by Eakman et al. [1 S1 was
applied to the SCF process. In arder to simulate the process, the balance equation was
coupled to the limiting substrate, and DO balance.

The resulting set of equations

consisted of a non-linear, panial-integro differential equation coupled to two differential

equations. A numerical scheme wu developed and used to solve the model for the SCF
process. A FORTRAN program was written, and is included in the Appendix. The
population balance equation as proposed by Eakman et al [IS] was able to capture the

97

biomass concentration, the limiting substrate concentration, and the DO concentration

profiles. These simulation outputs are quantitative measures which can be measured
directiy in the laboratory. However, il synchrony wu not captured by this model.

By introducing a feedbaek mechanism between the eritical division mass D1e and
the limiting substrate concentration

c.,

the eell mass model was able ta prediet eell

synehrony, along with the cell number profile. This mechanism was necessary in arder to
converge the microorganisms inta a common cell mass, and overcome the effect of
dispersion inherent in a replicating cell culture.

98

~ter 4.

Conclusions

The general objective ofthis work was to formulate a microbial population balance
mode!, and use it to simulate the SCF process. The approach taken wu to fonnulate a
model, develop a solution scheme to solve it, and validate the model usmg existing
experlmental data and theoretical results from previous modeling work. Two different
segregated population balance models of SCF were formulated: a cell age population
balance model and a cell mass population balance model.
Ta model SCF using the cell age model, the ceU age population balance equation
was solved for an osci11ating division age. These oscillations in the division age occur due
ta oscillations in the growth rate, which arise in SCF because of the oscillations in the
Iimiting substrate concentration. These oscillations are major characteristics of the SCF
proeess, and result fram the periodie cycling of the system. The results of this model
showed that although the ceU age model predicted eeU synehrony in the SCF proeess, onee
the system settled itself into a mode of repeatable cycles, the resulting eeU density profile
did not reproduce what is observed in experiments. The madel predieted a step increase

in the total cell number at the beginning of each SCF cycle. Experimental data suggest
that this step increase in cell number occurs at the end ofeach SCF cycle. It was therefore
eoncluded that a ceU division model govemed strietly by cclI age is not adequate to
simulate a cell culture where the growth rate is not constant.
In the case of the cell mass model, the cell mass population balance equation, as
described by Eakman et al., was coupled to a limiting substrate balance and a DO balance.
The mus balance on the DO concentration was necessary since the DO concentration was

99

the parameter monitored to control cycling. This resulted in a system of equations

consisting of a non...linear, partial-integro differential equation, coupled to two non-lnear
differential equations. The equations were also modified to account for the filling and
emptying of the reactor. An efficient numerical scheme was develop ta solve this system
of equations, and provided oscillation ftee solutions. In addition, the implicit predictorcorrector Euler time integration method proved to be an efficient scheme to solve this
system of equations as a function of time, and was shown to handle the time
discontinuities without difficulty.
The cell mass model was successful in capturing the macroscopie features of the
system. A comparison of the predicted trends with the trends predieted by the model
developed by Wincure et al. showed that the biomass concentration, the limiting substrate
concentration and the DO concentration profiles were successfully captured by the new
modeL However, this cell mass model was not able capture the feature of cell sYnchrony.
A feedback mechanism between the critical division mass and the limiting substrate
concentration was incorporated iota the modeL The functionality of the division mass is
thoroughly what has been observed in experimental work. The resulting Madel predieted
cell number profiles similar ta those measured during SCF experiments. However, precise
and quantitative validations of the model remain to be done.

This will require the

measurement of cell mass distnDutions aloog with studies on the relationship between the
division mass and the substrate concentration.
Further investigation of the model could give greater insight ioto the behavior of
the system. More specifically, the effect of the filVempty ration on the cell population

100

could be studied. For example, how would the cell mass distribution redistribute itself if
the fill fraction was moved away from 1/27

Such studies could lead to a better

understanding of tms fermentation process, and of the synchronization phenomenon in

certain cell cultures.

101

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lOS

APPENDIXI

C THIS PROGRAM WAS WRlTIEN BY FRANCOIS OODIN TO SOLVE

C mE POPULATION BALANCE EQUATION COUPLED TO mE LIMlTING
C SUBSTRATE AND THE DISSOLVED OXYGEN BALANCES FOR mE CASE OF THE
C BATCH, CHEMOSTAT, AND SELF-CYCLING FERMENTATION PROCESS.
C

IMPLIClT REAL*8 (A-R. O-Z)

DIMENSION W(3), OP(3). X(61). CI"(63), CT0(63), CTOO(63)
DIMENSION CTP(63). SF(63). SK(63,63). 01(63). PHI(2)
DIMENSION PHIX(2). INDX(63)
C - DEFINING THE MESH
NE=60

NP=NE+3
RMESH=1.3DO
DO 21=1.10
2
X(f)=OFLOATO-l)/DFLOAT(NE)*2.0DO
DO 3 1=11,31
3
X(I)=X(l0)+OFLOATO-I0)*0.01DO
DO 4 I=32,NE+ 1
4
X(I)=DFLOATO-l)/DFLOAT(NE)
C - INPUT DATA FOR GAUSS QUADRATURE
DATA W /0.277777778oo,O.44444444444DO,0.277777778oo/
DATA GP/0.11270166S4DO,O.SDO,O.8872983346DO/
RICLA=14S.2DO
T=O.ODO
NCYCLE=l
02=10.000
C - DEFINE PARAMETRIC DATA
TP=O.ODO
TPl=O.ODO
0TP=2.0DO
PI=3.141592653600
ERRORL=l.OD-6
MAXK=30
MAXCYCLE=lO
RCS=O.lDO
RNO=l.OD24
R=S.OD-oS
XC=3.0D12
RKS=O.OOOOlDO
UC=O.ODO
PHIM=1.5lSD-oS
B=1.31DO
E=3.0DO/4.000*DSQRT{2.0DO)*1.OD-13
El=O.SD-13
RHO=l.OlDO
CSO=O.6246DO
nTA=l.ODlOO
02SAT=O.0371DO
YOS=S.4SDO
REY=l.ODO
C - DEFINE INITIAL CONDmONS
ICOONT=S3
OPEN (lCOUNT)

ICOONT=52
OPEN (lCOUNT)
ICOUNT=Sl
OPEN (lCOUNT)
ICOUNT=SO
OPEN (lCOUNT)
006 J=l,NE+l
cr(J)=O.OOIDO(1.ODO-(4.000(X(J)'().6SDO4.0DO)
IF (cr(J)LT.ERRORL) cr(J)=O.ODO
CTO(J)=Cf(J)
6
CI'OO(J)=CTO(I)
cr(NE+l)=O.OOO
crO(NE+l)=CT(NE+ 1)

CTOO(NE+l)=cro(NE+1)
CI'(NP-I)=2.0DO
crO(NP-l)=2.0DO

crOO(NP-l)=2.000
CT(NP)=O.2624DO
CTO(NP)=O.2624DO
CTOO(NP)=O.2624DO
C - PREDICTOR STEP WHEN CONVERGED SOLUTION HAS SATISFIED PROPERTIES
DT=o.omo
DTO=O.02DO
EPS=o.OOO 100
148 DO 4S K=I,NP
CTP(K)=Cf(K)+DTCT(K)-CrO(KIDTO)

crOO(K)=CTO(K)
CTO(K)=CT(K)

4S
cr(K)=CTP(K)
C-ASSEMBLY
C -INITIALIZE NEWfON-RAPHSON ITERATION COONTER.
60

JK=O

C -INITIALIZE SK=Q AND SF=O

64 DO 35 N=l,NP
SF(N)=O.ODO
DO 3S Nl=l,NP
35
SK(N,Nl}=O.ODO
C - LOOP OVER nIE ELEMENTS
JK=JK+l
DO 100 I=l,NE
DX=X(I+I)-X(l)
C - LOOP OVER THE GAUSS POINTS
DO 100 1=1,3
CALL TFUNCT(GP(J),Dx.PHI.PHIX)
CON=O.ODO
CONX=O.ODO
CONO=O.ODO
DO 90 L=I,2
Ll=I+L-1
CON=CON+PID(L) *cr(L1)
CONX=CONX+PIUX(L)cr(LI)
90
CONO=CONO+PBI(L).CTO(LI)
DCON=(CON-eONO)IDT
XON=X(l)+GP(J)DX

C - LOOP OVER 'IllE BASIS FUNCllONS

DO l00L=I,2
Ll=I+L-1
SF(LI)=SF(Ll)+DX*W(I}*(DCON+
RDRMCSDM(XON,CT(NP-I),PHlM,RMESH,XC,RKS,R,RHO,UC)*CON/(RMESH*XC)+
RRMCS(XON,CT(NP-l),PHIM,RME5aXC,RKS,R,RHO,UC)*CONXJ(RMESH*XC)R2.0DO*RINTEG1(XON,x,r,NE,CT,RMESH,XC,CT(NP-l),PHIM,RKS,R,RHO,UC,
RE,El,PI,GP(J),W,GP)+(GMCS(XON,Cf(NP-I),PHlM,RMESH,XC,RKS,RCS,R,RHO
R,UC,E,PO+1.ODOrrHETA)*CON)*PHI(L)

SK(Ll,NP-I)=SK(Ll,NP-I)-DX*W(J)*(CON*
RDDRDMDC(CT(NP-l),PHIM,RMESH,XC,RKS,R,RHO,UC)/(RMESH*XC)+CONX*
RDRMCSDCS(XON,CT(NP-I),PHIM,RMESH,XC,RKS,R,RHO)/(RMESH*XC)..2.0DO*
RDIIDCS(XON.X.I.NE,CT,RMESH,Xc,cr(NP-l},PHIM,RKS,R,IO,E,E l,PI
~OP(J).W,CiP)+CONDGMCSDCS(XON,CT(NP-l),PHIM,RMESH,XC,RKS,RCS,R,RHO

R.E.PO>PHI(L)
DO 100 M=1.2
MI-I+M-l
SK(Ll.MI)=SK(LI,MI)-W(I}*OX*(pID(M)IDT+PHI(M)*
RDRMCSDM(XON.CT(NP-I),PHIM,RMESH,XC,RKS,~RHO,UC)/(RMESH*XC)+

RRMCS(XON.Cf(NP-l),PHIM,RMESH,XC,RKS,R,RHO,UC)*PHIX(M)/(RMESH*XC)
R-2.000 e OII DW(XON)C.I,M,NE,CT,RMESH,XC,CI'(NP-I),PHIM,RKS,R,RHO
R,UC.E.El.PI.GP(J}.W,GP)+(GMCS(XON,CT(NP-I),PHIM,RMESH,XC,RKS,
RRCSARHO.UC.E.PI)+l.ODOrrHETA)*PIn(M*PHI(L)
100 CONTINUE
DCON-(CT(NP-l )-CTO(NP-lIDT
SF(NP-l)-OCON-(CSO-CT(NP-IrrHETA+
RRlNTE02(X.~CT.RMESH,XC,cr(NP-I),PHIM,RKS.B,R,RHO,RNO, W,GP)
DO 1051-1.NE+l
SK(NP-l.I)=-DI2DW(XJ.NE,RMESH,XC,CT(NP-l)'pHIM,RKS,B,R,
RRHO.RNO.W,GP)
lOS CONTINUE
SK(NP-l,NP-I)=-(l.OnOIDT+I.ODOrrHETA+
RDI2DCS(xNE.CT,RMESH,XC,CT(NP-l),PHIM,RKS,B,R,RHO,RNO,W,GP})
DCONl =(CI"(NP)-crO(NPIDT
SF(NP)=OCON1-(02SAT-eT(NPtrHETA-RKLA(02SAT-CT(NP+YOS
RRINTEG2OCNE,CT,RMESH,XC,CT(NP-l),PHIM,RKS,B,R,RHO,RNO,W,GP)
00 106 1= I,NE+ 1
SK(NP,n=-YOSDI2DW(X,I,NE,RMESH,XC,cr(NPl),PHIM,RKS~,

RRJUlO,RNO,W,GP)
106 CONTINUE
SK(NP,NP-l)=-(YOSOI2DCS(X,NE,CT,RMESH,XC,CT(NP-I),PHlM,RKS,B,R,
RRHO,RNO,W,GP
SK(NP,NP)=-1.0DOIDT-l.ODOrrHETA-RKLA
C - APPLY BOUNDARY CONDmON
SF(l)=O.ODO
SF(NE+1)=0.000
DO 108 1=l,NP
SK(l,I)=O.ODO
lOS
SK(NE+1,1)=0.000
SK(1,l)=l.ODO
SK(NE+l,NE+l)=l.ODO
C - CALL THE MATRIX EQUATION SOLVER ROUTINES
CALL LUDCMP(SK,NP,NP,INDX,D)
CALL LUBKSB(SK,NP,NP,INDx,SF)

DO 110 I=I,NP
110

CT(I)=Cf(l)+SF(1)
IF (CI'{NP-l)LT.l.0n-oS) REY=O.ODO
IF (CI'{NP-l)LT.O.ODO) 1BEN
PRINT *, 'CS LESS mAN ZERO'
CLOSE (lCOUNT)
ICOUNT=ICOUNT+l
CLOSE (lCOUNT)

ICOUNT=ICOUNT+1
CLOSE (lCOUNT)
ICOUNT=ICOUNT+1
CLOSE (lCOUNT)
STOP
ENDIF
C - CALCULATE THE EUCLIDEAN NORM OF nc
ERROR=O.ODO
DO 120 1=l,NP
120 ERROR=ERROR+SF(I)**2
EUCLIN=DSQRT(ERROR)
C-ERROR
IF (EUCLIN.LT.ERRORL) GOTO ISO
C -lTERATION COUNTER
IF (JK.GT.MAXK) OOTO 130
00T064
C - MESSAGE OF NON CONVERGENCE
130 WRITE(lCOUNT,140)'PROGRAM Dm NOT CONVERGE IN ',MAXK,' lTERATIONS'
WRlTE(ICOUNT, 141)'USING A',NE,' ELEMENT MESH'
140 FORMAT(A28,IJ,AI9)
141 FORMAT(A7,IJ,AI3)
CLOSE (lCOUNT)
ICOUNT=ICOUNT+1
CLOSE (lCOUNT)
ICOUNT=ICOUNT+ 1
CLOSE (lCOUNT)
ICOUNT=ICOUNT+l
CLOSE (lCOUNT)
STOP
ISO IF (JK.GT.3) EPS=EPS*O.SDO
IF (JK.EQ.3) EPS=EPS
IF (JK.LT.3) EPS=1.2*EPS
C - CALCULATE mE ERROR NORM
DO lIK=l,NP
18
DI(K)=(Cf(K)-CI'P(K))I2.0DO

CMAX=O.ODO
DNORMl=O.ODO
DO 19 KA=I,NE+l
IF (Cf(KA).GT.CMAX) CMAX=CT(KA)

DNORMI=DNORMI+DI(KA)**2
19

CONTINUE
CSMAX=cr(NP-I)
02MAX=CT(NP)

DN0RM2=DI(NP-I)**2

DNORM3-DI(NP)**2
DNORM=DSQRTDNORM2l(CSMAX**2)+(DNORMII{CMAX**2/(NE+1)

R+REY(DN0RM3/(02MAX2)

C - CALCULATE NEW TIME STEP

DTN=DT*DSQRT{EPSIDNORM)
C - TIME STEP CONTROL
IF (DTN.GEDT) OOTO 93
ADT=O.800*DT
IF (DTN.GE.ADT) ooro 94

DT=DTI2.0DO
OOTO 149
93

DTOO=DTO

DTO=DT
DT=DTN
OOTO 198
94 DTOO=DTO
DTO=DT
DT=DT
198 CONTINUE
C-TIME
T=T+DTO
PRINT ., JIe: ',NCYCLE
WRITE(lCOUNT, 199) T,CT(NP-l),cr(NP)
199 FORMAT(3EI2.6)
CALLRINTEG34(RINTEG3~G4.x,NE,N,CT,RMESH.XC,RNO,W,GP)

ICOUNT=ICOUNT+1
WRITE(lCOUNT, 400) T~G3/(ERNO)
400 FORMAT(2EI2.6)

ICOUNT=ICOUNT+l
WRITE(lCOUNT, 410) T.RINTEG4
410 FORMAT(2E12.6)

C
C
C
C

IF (T.GT.1Fl).AND.(NTP.LT.4 1HEN
ICOUNT=ICOUNT+l
DO 200 I=l,NE+l
WRlTE(lCOUNT, 300) X(I),cr(l)

C300
C200

C
C

C
C
C

FORMAT(2E12.6)
CONTINUE

TPl=TPl+DTPI3.0DO

ICOUNT=ICOUNT-3
NI'P=NTP+1
ELSE
ICOUNT=ICOUNT-2
ENDIF
IF (Cf(NP).GT.02) mEN

ICOUNT=ICOUNT+3
DO 490 I=l,NE+l
WRlTE(lCOUNT, 495) X(l),CT(I)
495
490

FORMAT(2EI2.6)
CONTINUE

ICOUNT=ICOUNT3
DO 450 I=I,NE+l
450
CI'(I)-CI'(I)12.0DO
Cf(NE+2)=cr(NE+2)12.0DO+CS0I2.0DO
Cf(NP)=Cf(NP)12.0DO-t025ATI2.0DO
NCYCLEcNCYCLE+l
02=100.000

DTP=TTP
IF (NCYCLE.EQ.2) DTP=O.3DO
TP=T
NTP=1

ENDIF
IF (Cf(NP).LT.02) 02=cr(NP)
C - TIME CONTROL
IF (NCYCLE.GT.MAXCYCLE) THEN
PRINT * 'MAXCYCLEt
CLOSE (lCOUNT)
ICOUN'f=ICOUNT+1
CLOSE (lCOUNT)
ICOUNT=ICOUNT+l
CLOSE (ICOUNT)
ICOUNT=ICOUNT+ 1
CLOSE (lCOUNT)
STOP
ENDIF
t

OOTO 148

C - PREDICfOR STEP WHEN CONVERGED SOLUTION IS RETURNED DUE TO A LARGE

C-TIME
149 DO 113 K=I.NP
crP(K)=crO(K)+D~crO(K)-CTOO(KIDTO)

113

c
C

c
C

c
C

CT(K)=CTP(K)
GOT060

END
FUNCTION RMCS(X.CS,PHIM,RMESH,XC,RKS,R,RHO.UC)
RETIJRNS mE SINGLE LL MASS GROWTH RATE
IMPLICIT REAL*& (A.H. OZ)
RMCS=2.0DO*PIDMCS*RMESH*XC*XI(R*RHO*(RKS+CSUC*RMESH*XC*X
RETIJRN

END
FUNCTION DRMCSDM<X,CS,PHIM,RMESH.XC,RKS,RJUIO,UC)
RETIJRNS THE FlRST DERIVATIVE OF mE SINGLE CELL RATE OF MASS INCREASE
IMPLICIT REAL*& (AH. Q.Z)
DRMCSDM=2.000*PHIM*CS*RMESH*XCI(R*RHO*(RKS+CSUC*RMESH*XC
RETIJRN
END
FUNC1'ION GMCS(X.CS,PHIM.RMESH.Xc,RKS,RCS,RJUIO,UC.E'p1)
RETIJRNS nIE PROBABIL1TY DMSION OF CELL OF MASS M
IMPLIClT REAL*& (A.II. Q.Z)
REY=l.ODO
IF (RMCS(X.CS.PHIM.RME5H.XC.RKSRHO.UC).LT.O.ODO) REY=O.ODO
GMCS=2.000*DEXP(-(RMESH*XC*XRMC(CS,RCSIE)2)!
R(E*(PI)**O.SOO*ERFC(RMESH*XC*X..RMC(CS,RCSIE
RRMCS(X.CS.PHIM.RMESH.XC,RICS,R,RHO,UC)*REY
RETIJRN
END

FUNCI10N RMC(CS,RCS)
RETIJRNS THE MEANDMSION MASS

IMPUClT REAL8 (A-H, O-Z)

IF (CS.GT.O.ISDO) mEN
RMC=3.8D-12
ELSE IF CS.LE.O.IIDO).AND.(CS.GE.o.omO THEN
RMC=3.8D-12..Q.8D-12l0.l300*(0.lIDO-eS)
ELSE
RMC=3.0D-12
ENDIF
REnJRN
END

FUNcrION RINTEGl{XON,X,I,NE,CI',RMESH)CC,CS,PHIM,RKS,R,RHO,UC
R.E,EI,PI,OPJ,W,GP)
C 1TIJRNS mE CELL BIRTIi TERM:
IMPLICIT REAL*8 (A..H. O-Z) .
DIMENSION X(6l), CT(63), W(3), OP(3), PHI(2), PHIX(2)
RINTEGl=O.ODO
DXl=X(I+l)-XON
DX=X(I+l}-X(I)
DO 1000 J=I,3
CALL TFUNCTGPI+GP(J)*(l.ODO-GPI),Dx,pln,PHIX)
CONl=O.ODO
DO lOlO L=I,2
Ll=I+Ll
lOlO
CONI=CON1+PHl(L)*CT(L1)
XONI=XON+GP(J)*DXl
RINTEGI=RINTEGI+W(J)*DXI
RGMCS(XONI,CS,PHIM,RMESH,XC,RKS,RCS,R,RHO,UC.E'p1)
R*PMMl(XON,XONl,RMESH,XC.EI,PI)*CONIRMESHXC
1000 CONTINUE
DO 1020 ID=I+l,NE
DX=X(ID+ l)X(ID)
DO 1020 1=1,3
CALI.. TFUNCI'(OP(J),Dx,pHI,PHIX)
CON1=O.ODO
DO 1030 L=I,2
Ll=ID+L-l
1030
CONI-CONI+PJn(L) *CT(L1)
XONI=X(ID)+GP(J)DX
RINTEG l-RINTEGI+W(I)*DX*GMCS(XONI,CS,PHIM,RMESH,
RXC,RKS,RCS.R.RHO,UC,E,PI)*PMMI(XON,XONI,RMESH,XC,EI,PI)
RCONl*RMESH*XC
1020 CONTINUE
REnJRN

c
C

END
FUNcrION PMMI(X,Xl,RMESH,XC,El,P1)
RETIJRNS nIE PROBABILlTY OF CELL BIRTH OF MASS M FROM A LL OF MASS M'
IMPUCIT REAL8 (AR, Q.Z)
PMMI=DEXP(-X-X IJ2.0DO)RMESHXCIE1)**2)/
R(EI*PI*0.SOO(l.OOOERFC(XI*RMESH*XCI(2.000*EI
REnJRN
END

FUNCl'ION RINTEG2(X,NE,cr,RMESH,XC,CS.flDM,RK8,sRHO,RNO,W,GP)
RE1lJRNS THE RATE OF SUBS1RATE CONSUMPTION
IMPLICITREALS (A-H, O-Z)
DIMENSION X(61), CT(63), W(3), OP(3), PIn(2), PHIX(2)
RlNTEG2=O.ODO
DO 1040 1=l,NE
DX=X(I+l)-X(I)
DO 1040 1= 1,3
CALL TFUNCI'(GP(J),Dx.PHLPHIX)
CON1=O.ODO
DO 1060 Lz:l,2
Ll=I+L-l
1060
CONl=CONl+PHI(L)cr(Ll)
XONI=X(I)+GP(J)*DX
RINTEG2=RINTEG2+W(J)*DX
RBRIMCS(XONl,CS'pHIMJUdESH,XC,RKS,IUUlO)*RNO*CONI*RMESH*XC
1040 CONTINUE
RE1lJRN

END
C

FUNCTION RIMCS(X,CS,PHIM,RME8H,XC,RKS,IUUlO)
RE1lJRNS THE SINGLE CELL RATE OF MASS INCREASE
IMPLIClT REAL*8 (A-II, O-Z)
RI MCS=2.0DO*PHIM.CS*RMESH*XC*XI(R*RHO.(RKS+CS
RE1lJRN
END
FUNCTION DIlDW(XONXI~NE,CT;wESH,XC,
RCS,P~RKS,R,RHO,UC,E,El,PI,GPI,

W,GP)

RE1lJRNS THE DIRIVATIVE Of niE CELL BIRTH TERM wrm RESPECT TO WJ

IMPLICIT REAL8 (A-II, Q-Z)
DIMENSION X(61), CT(6J), W(J), OP(3), Plfi(2), PHIX(2)
DIlDW=O.ODO
DXl=X(I+l)-XON
DX=X(I+I)-X(I)
DO 1070 1= 1,3
CALL TFUNCfGPI+GP(J)*(l.ODO-GPJ),DK,PHI,PHIX)
XON1=XON+GP(J)*DXl
DIIDW=DIlDW+W(J)DXlGMCS(XONl,CS,PHIM,RMESH,XC,RKS,RCS,R,

RRHO,UC~'pI)PMMl(XON,XONl;wESH,XC~I'pI)PID(M)RMESH*XC

1070 CONTINUE
IF M.EQ.2).ANO.(I.LT.NE TIIEN
DX=X(I+2)-X(I+l)
DO 1080 J:;I,3
CALL TFUNCT(GP(J),Dx,FHI,P1DX)
XONt=X(I+I)+GP(1)*DX
DI1DW=DI1DW+W(J)DX*GMCS(XONI,CS,PHIMJUdESH,XC.RKS,RCS,R,
RRHO,UC.E,PI)*PMMI(XON,xONI;wESH,XC,EI,PI)*PHI(I)RMESHXC
1080 CONTINUE
ENDIF
RE1lJRN
END

FUNCI'lON DDRDMDC(CS,PHIM,RMESH,XCJUCS,ll,RHO,UC)

REnJRNS nIE DERIVATIVE OF DRMCSDM wrm RESPECT TO CS

IMPUCIT REAL*S (A-a. Q-Z)
DDRDMDC=2.0DO*PHIM*RMESH*XC*RKS/(RRHO*(RKS+CS)**2)
REnJRN
END

FUNCTION DRMCSDCS(X,CS,PHlM,RMESH,XC,RKS,IUUlO)
REnJRNS 1HE DERIVATIVE OF RMCS WIH RESPECT TO CS
IMPUClT REAL8 (A-a o..l)
DRMCSDCS=2.000*PHIM*RMESH*XC*X*RKSI(RRHO(RKS+CS)*2)
REnJRN
END

FONCTION DIlDCS(XON,X;,NE,Cf,RMESH,XC,CS,PHIM,
RRKS,IUUlO,E,EI,PI,GPI,W,OP)
C REnJRNS 1HE DERIVATIVE OF n CELL BIRTH TERM WITH RESPECT TO CS
IMPLIClT REAL*8 (A-H, Q-Z)
DIMENSION X(61), CT(63), W(3), OP(3), PHI(2), Pmx(2)
DIlDCS=O.ODO
DXI=X(I+l)-XON
DX=X(1+1)-X(I)
DO 10901=1,3
CALL TFUNCTCCOPJ+GP(J)*Cl.ODO-opJ),Dx,pm,pHIX)
CONI=O.ODO
DO 1100 L=I,2
LI=[+L-l
1100
CON1=CONI+PHI(L)cr(L1)
XONI=XON+<iP(J)*OXI
DIlDCS=DIlDCS+W{J)OX1*DOMCSDCS(XON1,CS,PHIM,RMESH,
RXCJU{S,RCS,R,RHO,UC.E,PI)*PMMI(XON,XONI,RMESH..XC.El,PI)
RCONl*RMESH*XC
1090 CONTINUE
DO 1110 ID=I+I,NE
DX=X(lD+l)..X(ID)
DO 1110 J=I,3
CALL TFUNCI"(GP(J),DX,PHI,PHIX)
CON1=O.ODO
DO 1120 L=I,2
LI-ID+L-I
1120
CONI=CONI+PIU(L)*cr(L1)
XONI=X(ID)+GP(J)*DX
DIIDCS=DIIDCS+W(J)DX*DGMCSDCS(XONI,CS,PHIM,RMESH..
RXC,RKS,RCS,IUUlo,UC,E,PI)PMMI(XON,XONI,RMESH,XC,EI,P1)
RCONI*RMESHXC
IlIa CONTINUE
REnJRN
END
C

FUNCI'ION DGMCSDCS(X,CS,PHIM,RMESH,XC,RKS,RCS,IUUlO,UC,E,P1)
REnJRNS mE DERIVATIVE OF 1BE GAMMA FUNcrION WlTIIRESPEcr TO CS

IMPLIcrrREAL*s (A"1l Q-Z)

REY=l.ODO
IF (RMCS(X.CS,PHIM,RMESH,XC,RKS,RJUIO,UC)LT.O.ODO) REY=O.ODO
DGMCSDCs-2.000*DEXP(-(RMESH*XC*XRMC(CS,RCS)IE)*2)1(E(P1)

RO.5DOERFC({RMESH*XCX-RMC(CS,RCS)IE*
RDRMCSDCS(X.CS'pHIM,RMESH,XCJU{S-RRHO)REY+2.0DO/(E*DSQRT(Pl)*
R(DEXP(-({RMESH*XCX-RMC(CS,RCSIE)**2)2.000*
R{RMESHXC*X-RMC(CS,RCS)IE*2*ERFCRMESHXCX
R-RMC(CS,RCS)IE)*DMCDCS(CS,RCS)-DEXP(-({RMESHXC*XRRMC(CS,RCS)IE)2)*DERFCDCS(X,CS,RMESH,XC,RCS,E,PI)
RRMCS(X.CS,PHIM,RMESH,XC,RKS,RJUlO,UC)1
RERFC({RMESH*XCX-RMC(CS,RCSIE)**2
REnJRN
END
C
C

FUNCTION DMCDCS(CS,RCS)
REnJRNS nIE DERIVATIVE OF MC WlTIIRESPECTTO CS
IMPLICIT REAL8 (A-a O-Z)

IF (CS.GT.0.15DO) TIIEN
DMCDCS-o.ODO
ELSE IF CS.LE.O.lSDO).ANO.(CS.GE.O.02DO nN
DMCDCS=O.8D-12JO.13DO
ELSE
DMCDCS-O.ODO
ENDIF
REnJRN
END
C
FUNCTION DERFCDCS(X,CS,RMESH,XC,RCS,E,PI)

REnJRNS nIE ERFC FUNcrION DIFFERENTIATED Wl1H RESPECT TQ CS

IMPLICIT REAL*8 (A-R, O-Z)
DERFCDCS--2.0DOIDSQRT(pI)*DEXP(-({RMESH*XC*X-RMC(CS,RCS)IE)**2
R)*DMCDCS(CS,RCS)IE
REnJRN

END
C
FUNcrION DI2DW<X,I,NE,RMESH,XC,CS,PHIM,RKS,B.R,RHO.RNO,W,GP)

REnJRNS mE RATE OF SUBSTRATE CONSUMPTlON

IMPLICIT REAL8 (A-R, D-Z)
DIMENSIONX(61), W(3), OP(3), PHI(2), PIllX(2)
D12DW=O.ODO
IF (I.NE.l).AND.(I.NE.NE 1lIEN
DO 113010=1,2
DX=X(I+ID)-X(I+ID-l)
DO 1130 J=I,3
CALL TFUNCf(GP(J),Dx,pm,PHlX)
XONI=X(I+m-l)+(ip(J)*DX
DI2DW=DI2DW+W(J)DX
RB*RIMCS(XON1,CSJ-HIM,RMESH,XC,RKS,RJUIO)RNO*PHI(3-1D)*RMESH*XC
1130 CONTINUE
ELSEIF (1.EQ.l) THEN
DX=X(I+I)-X(I)
DO 1140 1=1,3

CALL TFUNcr{GP(J).ox,pm,PHlX)

XONI=X(I)+GP(J)*DX
DI2DW=DI2DW+W(J)DX*
RBRIMCS(XONI,CS,PHIM,RMESH,XC,RKS,RJUIO)RNO*PIn(I)RMESH*XC
1140 CONTINUE

ELSE
DX=X(I+l)-X(1)
DO 1150 1=1,3
CALL TFUNCI'(GP(J),oK,PHI.PHIX)
XONI=X(l)+GP(J)*DX
DI2DW=DI2DW+W(J)*DX*
RB*RIMCS(XONI,CS,PHIM,RMESH,XC,RKS,RJUlO)*RNO*PHI(2)*RMESH*XC
1150 CONTINUE

c
C

ENDIF
RETURN
END
FUNCTION DI2DCS<X,NE,cr,RMESllxC,CS,PHIM,RKS,B,RRHO,RNO,W,GP)
RETURNS mE DERIVATIVE OF mE RATE OF SUBSTRATE CONSUI\1PTION
WInI RESPECf TO CS
IMPLICrr REAL *8 (A-H, O-Z)
DIMENSION X(61), cr(63), W(3), GP(3), PHI(2), PHIX(2)
DI2DCS=O.ODO
DO 1040 1=l,NE
DX=X(I+1)-X(1)
DO 10401=1.3
CALL TFUNcr(GP(J),Dx,FHI,PHIX)

CON1=O.ODO
DO 1060 L=I,2
LI=I+L-l
1060
CONI=CONI+PHI(L)* cr(L1)
XONl=X(I)+GP(J)*DX
DI2DCS=Dl2DCS+W(J)*DX

RB*DRIMCSDC(XONI,CS,PHIM,RMESH,XC,RKS,R.,RHO)*RNOCONI*RMESH*XC
1040 CONTINUE
RETURN
END

C
C

c
C

FONCTION ORIMCSDC<X,CS,PHlM,RMESH,XC,RKS,R,RHO)
RETURNS THE DERIVATIVE OF RIMes WJH RESPECf TO CS
IMPLIClT REAL*8 (A-R, O-Z)
DR 1MCSOC=DRMCSDCS(X,CS,PHlM,RMESH,XC,RKS,R.,RHO)
RETURN
END
SUBROtmNE RINTEG34(RINTEG3,RINTEG4,x,NE,N,Cf,RMESH,XC.RNO, W,GP)
RETURNS THE CELL DENSlTY AND nIE BIOMASS CONCENTRATION
IMPLIClT REAL*8 (A-H, O-Z)
DIMENSION X(NE), cr(N). W(3), GP(3), PHI(2), PIDX(2)
RINTEG3=O.ODO
RINTEG4=O.ODO
DO 1280 1=l,NE
DX=X(I+1)-X(1)
DO 1280 J= 1,3
CALL TFUNcr(GP(J),Dx,Fm,PHIX)
CONI=O.ODO
DO 1290 L-l,2
Ll=I+L-1

1290

CONI=-CONI+PlD(L)cr(LI)

XONl=X(l)+GP(J)*DX
RINTEG3=RINTEG3+W(J)*DX*CONl*RMESH*XC
RINTEG4=RINTEG4+W(J)*DX*CONl*XONl(RMESH*XC)**2
1280 CONTINUE
RINTEG3=RNO*RINTEG3
RINTEG4-RNO*RINTEG4
RETURN
END

FUNCI'ION ERFC(X)
C RETURNS THE COMPLEMENTARY ERROR FUNCl'ION ERFC(X)
IMPLICIT REAL*8 (A-R. Q-Z)
IF(X.LT.O.ODO)THEN
ERFC=1.OnO+GAMMP(O.SDO t X2)

ELSE
ERFC=GAMMQ(O.moX**2)
ENDIF
RETURN

c
C

END

FUNcrION GAMMP(A,X)
RETURNS n INCOMPLETE GAMMA FUNCTION P(A.X)
IMPUCIT REAL*8 (A-H, O-Z)
IF(X.LT.O.ODO.OR.A.LE.O.ODO)PAUSE
IF(X.LT.A+l.ODO}THEN
CALL GSER(GAMSER,A,K.GLN)
GAMMP=GAMSER

ELSE
CALL GCF(GAMMCFtA,X,OLN)

GAMMP=1.OOO-GAMMCF
ENDIF
RETURN

c
C

END

FUNcrION GAMMQ(A,X)
RETURNS nIE INCOMPLETE GAMMA FONCTION Q(A.X)= I-P(A,X)
IMPLIClT REAL*8 (A-H, o-Z)
IF(X.LT.O.ODO.OR.A.LE.O.ODO)PAUSE
IF(X.LT.A+1.ODO)nN
CALL GSER(GAMSER,A,K.GLN)
GAMMQ=l.ODO-GAMSER

ELSE
CALL GCF(GAMMCF,A,X.GLN)

GAMMQ=GAMMCF
ENDIF
RETURN
END
C

C
C

FUNCI10N GAMMLN(XX)
RETURNS n VALUE LN(GAMMA(XX FOR XX > O. FULL ACCURACY IS
OBTAINEDFORXX> 1. FORO<XX<l.
REAL8 COf(6),STP.HALFtONE,FPF.x,TMP,SER.
DATA COf,STPn6.18009173DO,-86.50S32033DO,24.0140982200,
* . .1.231739S16DO,.120IS8003D-2,-.S36382D-S,2.5066282746SDOI

DATA HALF,ONE,FPF/O.5DO, l.ODO,S.SDOI

X=XX-oNE

TMP=X+FPF
TMP=(X+HALF)*DLOG(TMP)-TMP
SER=ONE
DO 31=1,6
X=X+ONE

SER.-SER+COF(J)IX
3

c
C
C

CONTINUE
GAMMLN=TMP+DLOG(STP*SER)
RETURN
END

SUBROUTINE GSER(GAMSER,A,X,GLN)
RETURNS mE INCOMPLETE GAMMA FUNCI10N P(A,X) EVALUATED BY!TS
REPRESENTATION AS GAMSER. ALSO RETURNS LNGAMMA(A) AS GLN
IMPLICIT REAL-8 (A-a Q-Z)
PARAMETER (lTMAX=lOO,EPS=3.0D..(7)
GLN=GAMMLN(A)
IF(X.LE.O.ODO)1HEN
IF(XLT.O.ODO)PAUSE
GAMSER=O.ODO

RETURN
ENDIF
AP=A
SUM=l.ODO/A

DEL=SUM
DO Il N=l,lTMAX
AP=AP+l.ODO
DEL=DEL-XIAP
SUM=SUM+DEL
IF(DABS(DEL).LT.DABS(SUM)*EPS)OO TO 1
II CONTINUE
PAUSE tA TOO LARGE, lTMAX TOO SMALL'
1 GAMSER=SUM-DEXP(-X+A-DLOG(X)-GLN)
RETURN
END

SUBROUTINE OCF(OAMMCF,A,X,OLN)
IMPLIClT REAL*8 (A-H, C-Z)
C RETURNS nIE INCOMPLETE GAMMA FUNcrION Q(A,X) EVALUATED BV!TS
C CONTINUED FRAcrION REPRESENTATION AS GAMMCF. ALSO REUTURNS
C LNGAMMA(A) AS OLN
PARAMETER (lTMAX=lOO,EPS=3.0D.Q7)
GLN=GAMMLN(A)
GOLD=O.ODO
AOz1.0DO
Al=X

BO=O.ODO
BI-1.0oo

FAC=l.ODO
00 11 N=l,lTMAX

AN=DFLOAT(N)
ANA=ANA

AO=(AI+AOANA)FAC
BO=(BI+BOANA)-FAC
ANF=AN*FAC
AI=XAO+ANF*AI
Bl=XBO+ANPBI
IF(AI.NE.O.ODO)1lIEN
FAC= 1.0001Al
G=BlFAC
IF(DABSG-GOLD)lG).LT.EPS)OO TO 2
GOLD=G
ENDIF
12

CONTINUE

PAUSE 'A TOO LARGE, ITMAX TOO SMALL'

2 GAMMCF=DEXP(-X+ADLOG(X)-QLN)G
RETIJRN

END
C

C SUBROUTINE IMPLEMENTING LINEAR BASIS FUNcrION

C
SUBROUTINE TFUNCT(GP,ox.PHI'pHIX)
IMPLICIT REAL 8 (AH, Q.Z)
DIMENSION PID(2). PInX(2)
PHI(I)=l.ODO-GP
PHI(2)=GP
PHIX(l)=-l.ODOIDX
PIDX(2)= 1.ODOIDX
RETIJRN
END

C SUBROUTINES TO SOLVE AX=C TYPE PROBLEMS

C
C

C
C
C

GIVEN ANNXNMATRIXA. WI1HPHYSICALDIMENSIONNP, THIS ROUTINE

REPLACES 1T SY 11 LUDECOMPOSISION OF A ROWWISE PERMUTATION OF
ITSELF

SUBROUTINE LUDCMP(A,N,NP.INDXJ
IMPLICIT REAL *8 (AH, Q.Z)
PARAMETER (NMAX=IOO,TINY=1.0D-20)
DIMENSION A(NPtNP),INDX(N),VV(NMAX)
0=1.000
DO 1060 1= I,N
AAMAX=O.ODO
DO 1050 1=I.N
IF (ASS(A(I,J.GT.AAMAX) AAMAX=-ABS(A(l.J
1050

CONTINUE

IF (MMAXEQ.O.ODO) PAUSE 'SINGULAR. MATRIX.'

VV(l)=1.0DO/AAMAX
1060 CONTINUE
DO 12301-1.N
IF Q.GT.I) mEN
DO lOlO 1-1)-1
SUM=A(IJ)

IF (I.Gr.l)11N

DO 10701{=1.Il

SUM=SUMA(I.K)A(K,J)
1070

CONTINUE

A(I,J)=SUM

ENDIF
1080

CONTINUE

ENDIF
AAMAX=O.ODO
DO 1200 I=J,N
SUM=A(LJ)
IF (J.GT.l)THEN
DO 1090 K=l,Il
SUM=SUM-A(LK)A(K,J)
1090
CONTINUE
A(I,J)=SUM

ENDIF
DUM=W{l)ABS(SUM)

IF (DUMGE.AAMAX) 1HEN
!MAX=I
AAMAX=DUM
ENDIF
CONTINUE
IF (I.NElMAX)nlEN
DO 1210 K=l,N
DUM=A(IMAX,K)
A(IMAX,K)=A(J.K)
A(J,K)=DUM
1210
CONTINUE
D=-D
VV(lMAX)=W(J)

1200

ENDIF
INDX(J)=IMAX

IF(J.NE.N)THEN
IF(A(I,J).EQ.O.ODO)A(I,J)--TlNY
DUM=l.ODO/A(I,J)
DO 1220I=I+l,N
A(I,J)=A(I,J)DUM
1220 CONTINUE

ENDIF
1230 CONTINUE
IF(A(N,N).EQ.O.ODO)A(N,N}--TlNY
RETURN

END

mIS ROUTINE SOLVES 11IE SET OF N LINEAR. EQUATIONS AX=B. HERE A

15 INPUT, NOT AS THE MATRlXBUTRATHER AS rrs LUDECOMPOSmON,

DETERMINED BY THE ROUTINE LUDCMP

SUSROUTINE LUBKSB(A.N,NP,INDU)
IMPLIClT REAL *8 (A-&, C-Z)
DIMENSION A(NP,NP),INDX(N),B(N)
IT=O
DO 1250 I=I,N
LL-INDX(1)

SUM=B(LL)
B(LL)=B(I)
IF (ILNE.O)nN
DO 1240 1=II,I-l

SUM=SUM-A(I,J)*B(J)
CONTINUE
ELSE IF (SUM.NE.O.ODO) 'mEN
n=I
ENDIF
B(l)=SUM
1250 CONTINUE
DO 1270 I=N,I,-1
SUM=B(I)
IF(I.LT.N)THEN
DO 1260 1=1+I,N
SUM=SUM-A(I,J)*B(J)
1260
CONTINUE

1240

ENDIF

B(I)=SUMIA(I,I)
1270 CONTINUE
RETURN

END